Compositions and methods for lyophilization of bacteria or listeria strains

ABSTRACT

Methods and compositions are provided for lyophilization of bacteria or Listeria strains, such as Listeria monocytogenes. Provided are methods for producing a lyophilized composition comprising a bacteria or Listeria strain, formulations for lyophilization comprising a bacteria or Listeria strain, lyophilized bacteria or Listeria strains, and methods of preparing frozen bacteria or Listeria strains for lyophilization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry of PCT/US2018/048586 filedAug. 29, 2018, which claims the benefit of U.S. Application No.62/560,318, filed Sep. 19, 2017, herein incorporated by reference in itsentirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The Sequence Listing written in file 545193SEQLST.TXT is 11,733,993bytes, was created on Mar. 17, 2020, and is hereby incorporated byreference.

BACKGROUND

Lyophilization is a process that removes solvent from a solution to formsolid or powder that is stable and easier to store at elevatedtemperature than liquid. Lyophilization, also known as freeze drying,involves freezing followed by sublimation. The resulting lyophilizedmatter may be stored without refrigeration or at higher temperaturesthan liquid, reducing storage and transportation costs of the substanceas well as the storage space required for the product. It also canreduce the weight of the product, which similarly reduces shipping andrelated costs. Lyophilization is particularly useful for preserving andstoring various biological molecules, because it increases theirshelf-life.

Compared to liquid formulations, solid formulations have multipleadvantages such as superior storage stability, reduced molecularmobility and unwanted chemical reactions, and less package weight forincreased ease in shipping and distribution. Furthermore, as allcurrently available commercial vaccines require low temperature storage,the goal is to utilize solid state stabilization techniques to enhancetheir room temperature or high temperature stability and to reduce thereliance on cold-chain to maintain efficacy and ensure safety. Althoughlyophilization is the preferred preservation method because the lowstorage and transport costs of freeze-dried bacterial cultures are amajor advantage compared to cryopreservation, lyophilization is a verycomplex physical process affected by many parameters requiring specificequipment and trained personnel. Freeze-drying can cause many types ofdamage to cells, including a loss of viability, reduction of metabolicactivity, and changes in cell morphology, which can affect thephysiology, characterization, and functions of cells such as bacteria.

In addition, the effect of varying different lyophilization parametersis highly strain-specific, and this strain dependency makes it difficultto draw general conclusions or guidelines from any one particularstrain. The high biological and metabolic diversity of bacteria makes itdifficult and laborious to develop strain-specific optimizedfreeze-drying procedures. There are very limited data on thelyophilization of Listeria bacteria strains such as Listeriamonocytogenes and what parameters need to be optimized and how tooptimize them to make lyophilization a viable option for Listeria.

SUMMARY

Methods and compositions are provided for lyophilization of bacteria orListeria strains, such as Listeria monocytogenes. In one aspect,provided are methods for producing a lyophilized composition comprisinga bacteria or Listeria strain. Some such methods can comprise providinga composition comprising a bacteria or Listeria strain in a formulationcomprising a buffer, cooling the composition in a freezing step,exposing the cooled composition to a vacuum and a first increasedtemperature in a primary drying step, and exposing the composition fromthe primary drying step to a vacuum and a second increased temperaturein a secondary drying step, whereby the lyophilized composition isproduced.

In some such methods, the bacteria or Listeria strain used in thecomposition is a frozen Listeria strain that is thawed prior to thefreezing step. In a specific example, the frozen bacteria or Listeriastrain can be thawed at a temperature of about 2° C. to about 37° C.,about 20° C. to about 37° C., about 23° C. to about 37° C., about 25° C.to about 37° C., about 32° C. to about 37° C., or about 37° C.Optionally, the thawing is for no more than about 8 hours. Optionally,the thawed bacteria or Listeria strain is held at temperature of betweenabout 2° C. and about 8° C. for no more than about 24 hours. In aspecific example, the concentration of the bacteria or Listeria strainbeing thawed can be between about 1×10E9 and about 1×10E10 colonyforming units (CFU) per milliliter.

In some such methods, the formulation comprises a buffer and sucrose.For example, the formulation buffer can comprise about 1% to about 5%w/v sucrose, about 2% to about 3% w/v sucrose, or about 2.5% w/vsucrose. Optionally, the formulation does not comprise one or more otherexcipients such as trehalose, monosodium glutamate (MSG), or recombinanthuman serum albumin (rHSA).

In some such methods, the formulation comprises about 1×10E9 to about1×10E10 colony forming units (CFU) of bacteria or Listeria permilliliter.

In some such methods, the holding temperature in the primary drying stepis between about −10° C. and about −30° C., between about −12° C. andabout −22° C., between about −17° C. and about −19° C., or about −18° C.

In some such methods, the residual moisture in the lyophilizedcomposition is at least about 2.5%, at least about 3%, or at least about3.5%. In some such methods, the residual moisture is between about 1%and about 5% or between about 2% and about 4%.

In some such methods, the lyophilized composition shows at least about60%, 70%, 80%, or 90% viability after storage at between about −20° C.and about 4° C. or after storage at about −20° C. or about 4° C. forabout 6 months, 12 months, 18 months, or 24 months.

Such methods can comprise, for example: (a) providing a compositioncomprising a Listeria strain in a formulation comprising a buffer andsucrose; (b) cooling the composition provided in step (a) to a holdingtemperature between about −32° C. and about −80° C. in a freezing step;(c) exposing the composition produced by step (b) to a vacuum at aholding temperature between about −10° C. and about −30° C. in a primarydrying step; and (d) exposing the composition produced by step (c) to avacuum at a holding temperature between about −5° C. and about 25° C. ina secondary drying step whereby the lyophilized composition is produced.Such methods can alternatively comprise, for example: (a) providing acomposition comprising a Listeria strain in a formulation comprising abuffer and sucrose; (b) cooling the composition provided in step (a) toa holding temperature between about −32° C. and about −80° C. in afreezing step; (c) exposing the composition produced by step (b) to avacuum at a holding temperature between about −10° C. and about −30° C.in a primary drying step; and (d) exposing the composition produced bystep (c) to a vacuum at a holding temperature between about 5° C. andabout 25° C. in a secondary drying step whereby the lyophilizedcomposition is produced. In some such methods, the Listeria strain is arecombinant Listeria monocytogenes strain, a stress response is inducedin the Listeria strain by exposing the Listeria strain to a decreasedtemperature, the buffer is a phosphate buffer, the formulation comprises2% to 3% w/v sucrose, the formulation does not comprise trehalose, MSG,or rHSA, the temperature in the primary drying step (c) is between −17°C. and −19° C., and the residual moisture in the lyophilized compositionis between 3% and 4%. Some such methods have one or more or all of thefollowing elements: the Listeria strain is a recombinant Listeriamonocytogenes strain; the buffer is a phosphate buffer; the formulationcomprises about 2% to about 3% w/v sucrose; the formulation does notcomprise trehalose, MSG, or rHSA; the formulation comprises about 1×10E9to about 1×10E10 colony forming units (CFU) of Listeria per milliliter;the holding temperature in the freezing step (a) is between about −40°C. and about −50° C.; the holding temperature in the primary drying step(c) is between about −17° C. and about −19° C.; the holding temperaturein the secondary drying step (d) is between −1° C. and 1° C.; and theresidual moisture in the lyophilized composition is between about 2.5%and about 4%. In some such methods, the Listeria strain used in thecomposition in step (a) is a frozen Listeria strain that is thawed priorto step (a). Optionally, such methods have one or more or all of thefollowing elements: the concentration of the frozen Listeria strainbeing thawed is between about 1×10E9 to about 1×10E10 colony formingunits (CFU) per milliliter; the frozen Listeria strain is thawed atabout 37° C.; the frozen Listeria strain is thawed for no more than 8hours; and the frozen Listeria strain is held at about 2° C. to about 8°C. for no more than 24 hours after thawing. Also provided arelyophilized bacteria or Listeria strains produced by the lyophilizationmethods disclosed herein.

In another aspect, provided are formulations for lyophilizationcomprising a bacteria or Listeria strain. Such formulations cancomprise, for example: (1) the Listeria strain; (2) a phosphate buffer;and (3) sucrose. In some such formulations, the Listeria strain is arecombinant Listeria monocytogenes strain, the formulation comprisesabout 2% to about 3% w/v sucrose, and the formulation does not comprisetrehalose, MSG, or rHSA.

In another aspect, provided are lyophilized compositions comprising abacteria or Listeria strain. Some such lyophilized compositions have aresidual moisture of at least about 2.5% or at least about 3%. Some suchlyophilized compositions can further comprise a phosphate buffer andsucrose. In some such lyophilized compositions, the Listeria strain is arecombinant Listeria monocytogenes strain, the lyophilized compositiondoes not comprise trehalose, MSG, or rHSA, and the residual moisture inthe lyophilized composition is between 3% and 4%.

In another aspect, provided are methods of preparing a frozen Listeriastrain for lyophilization, comprising thawing the frozen Listeria strainat a temperature between about 20° C. and about 37° C. Optionally, suchmethods have one or more or all of the following elements: theconcentration of the frozen Listeria strain being thawed is betweenabout 1×10E9 to about 1×10E10 colony forming units (CFU) per milliliter;the frozen Listeria strain is thawed at about 37° C.; the frozenListeria strain is thawed for no more than 8 hours; and the frozenListeria strain is held at about 2° C. to about 8° C. for no more than24 hours after thawing.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows viable cell count (VCC) data for different formulationbuffers (citrate, phosphate, and MOPS (3-(N-morpholino)propanesulfonicacid)) and for different ratios of sucrose (Suc) to trehalose (Treh) tomonosodium glutamate (MSG) to recombinant human serum albumin (rHSA)over time.

FIGS. 2A and 2B show VCC multivariate data analysis (MVDA) in anaccelerated stability study at a 1 month stability anchor point (FIG.2A) and at a 6 month stability anchor point (FIG. 2B).

FIG. 3 shows VCC data for different OD levels and stabilizercombinations. Heading for each plot indicates OD in vial (2.0, 3.0,10.0, 12.5, 15.0, 17.5, or 20.0), stabilizer (buffer 2 (2.5% sucrose) orbuffer 5 (5% sucrose with amino acid mix).

FIG. 4 shows VCC data for different OD levels and ratios of sucrose(Suc) to amino acid mix (AA Mix) to recombinant human serum albumin(rHSA). Heading for each plot indicates OD in vial (2 or 10), sucrose(10, 5, 2.5):amino acid mix (presence (1) or absence (0)):rHSA (0, 1, or2.5).

FIG. 5 shows residual moisture (RM) data for different OD levels andratios of sucrose (Suc) to amino acid mix (AA Mix) to recombinant humanserum albumin (rHSA).

FIG. 6 shows residual moisture data for different percentages of sucrose(weight per volume) at different time points in the lyophilizationcycle.

FIGS. 7A and 7B show VCC data post-lyophilization after storage atdifferent temperatures for different amounts of time in Lm samples afterthe primary drying step, after the ramp, or after the secondary dryingstep in the Lyo4 experiment. FIG. 7A shows results for formulationshaving 2.5% weight per volume (w/v) sucrose. FIG. 7B shows results forformulations having 5% w/v sucrose.

FIG. 8 shows VCC data post-lyophilization after storage at differenttemperatures for different amounts of time in Lm samples stressed with atemperature shift in an ice bath prior to lyophilization, Lm samplesstressed with acid treatment to lower pH prior to lyophilization, Lmsamples stressed with both a temperature shift and a pH shift prior tolyophilization, and Lm samples with no temperature shift or pH shift inthe Lyo5 experiment.

FIG. 9 shows VCC data (percent of average pre-lyophilization VCC) beforelyophilization (Initial) and post-lyophilization (all other samples)after storage at different temperatures for different amounts of time inthe Lyo6 experiment.

FIG. 10 shows VCC data (percent of average pre-lyophilization VCC)before lyophilization and post-lyophilization after storage at differenttemperatures for different amounts of time in the Lyo7 experiment.

FIG. 11 shows VCC data (percent of average pre-lyophilization VCC)post-lyophilization after storage at different temperatures fordifferent amounts of time for fresh Lm samples (Part A) and frozen Lmsamples that were thawed (Part B) in the Lyo8 experiment.

FIG. 12 shows VCC data post-lyophilization after storage at differenttemperatures for different amounts of time in the Lyo9 experiment.

FIG. 13 shows VCC data post-lyophilization after storage at differenttemperatures for different amounts of time in Lm samples stressed with atemperature shift in an ice bath prior to lyophilization (Part B) and Lmsamples with no temperature shift (Part A) in the Lyo10 experiment.

FIG. 14 shows VCC data post-lyophilization after storage at differenttemperatures for different amounts of time in the Lyo11 experiment.

FIG. 15 shows VCC data post-lyophilization after storage at differenttemperatures for different amounts of time in fresh Lm samples (Part A),frozen Lm samples thawed at 2-8° C. prior to lyophilization (Part B),and frozen Lm samples thawed at 37° C. and incubated 4 hours prior tolyophilization (Part C) in the Lyo12 experiment.

FIG. 16 shows VCC data post-lyophilization after storage at differenttemperatures for different amounts of time in fresh Lm samples (Part A)and Lm samples stored at 2-8° C. for 3 days.

FIG. 17 shows VCC data post-lyophilization after storage at differenttemperatures for different amounts of time in Lm samples under thefollowing conditions: 2R vials, 1×10⁹ VCC, and 1.2 mL fill.

FIG. 18 shows VCC data (CFU/mL) before lyophilization, afterlyophilization, and at accelerated conditions for 1, 2, and 3 days at30° C. (T_(liq), T_(lyo), T42 h, T48 h, and T72 h, respectively).

FIG. 19 shows residual moisture content (RM) as a function of the shelftemperature in the secondary drying step (SD temperature).

FIG. 20 shows the drug substance manufacturing process flow forAxalimogene Filolisbac (ADXS-HPV).

FIG. 21 shows a flow diagram for manufacture of the AxalimogeneFilolisbac (ADXS-HPV) drug product.

FIG. 22A shows VCC data (percent of average pre-lyophilization VCC)before lyophilization and post-lyophilization after storage at differenttemperatures for different amounts of time in the Lyo1 experiment.

FIG. 22B shows residual moisture immediately after lyophilization andafter 6 months at 2-8° C. in the Lyo1 experiment.

FIG. 23A shows VCC data (percent of average pre-lyophilization VCC)before lyophilization and post-lyophilization after storage at differenttemperatures for different amounts of time (months) in the Lyo2experiment.

FIG. 23B shows residual moisture immediately after lyophilization andafter 6 months at 2-8° C. in the Lyo2 experiment.

FIG. 24A shows residual moisture (RM) using 2.5% sucrose after primarydrying, after ramp, and after secondary drying after storage atdifferent temperatures for different amounts of time in the Lyo4experiment.

FIG. 24B shows residual moisture (RM) using 5.0% sucrose after primarydrying, after ramp, and after secondary drying after storage atdifferent temperatures for different amounts of time in the Lyo4experiment.

FIG. 25 shows residual moisture after various stress treatments afterstorage at different temperatures for different amounts of time in theLyo5 experiment.

FIG. 26 shows residual moisture following temperature shift treatmentpre-lyophilization after storage at different temperatures for differentamounts of time in the Lyo6 experiment.

FIG. 27 shows residual moisture after storage at different temperaturesfor different amounts of time in the Lyo7 experiment.

FIG. 28 shows residual moisture for samples were lyophilized immediately(part A) or samples that were frozen, thawed, and then lyophilized (partB) after storage at different temperatures for different amounts of timein the Lyo8 experiment.

FIG. 29 shows residual moisture after storage at different temperaturesfor different amounts of time in the Lyo9 experiment.

FIG. 30 shows residual moisture after storage at different temperaturesfor different amounts of time in the Lyo10 experiment.

FIG. 31 shows residual moisture after storage at different temperaturesfor different amounts of time in the Lyo11 experiment.

FIG. 32 shows residual moisture after storage at different temperaturesfor different amounts of time in the Lyo12 experiment.

FIG. 33 shows residual moisture after storage at different temperaturesfor different amounts of time in the Lyo13 experiment.

FIG. 34 shows residual moisture after storage at different temperaturesfor different amounts of time in the Lyo14 experiment.

FIG. 35 shows VCC data post-lyophilization as a percent ofpre-lyophilization after storage at 30° C. for different amounts of timein the batch scale experiment.

FIG. 36 shows VCC data post-lyophilization as a percent ofpost-lyophilization after storage at 30° C. for different amounts oftime in the batch scale experiment.

FIG. 37 shows residual moisture (RM) vs. sample in the batch scaleexperiment.

FIG. 38 shows residual moisture (RM) after lyophilization and storagefor 72 hours at 30° C. in the batch scale experiment.

FIG. 39 shows bioactivity and INFγ induction of the lyophilized productcompared to non-lyophilized bacteria and 10-mer control in the Lyo11experiment.

FIGS. 40A-B show VCC data (A) before lyophilization andpost-lyophilization immediately after lyophilization and after storageat 30° C. for 24, 48, or 72 hours, and residual moisture (B) in the WP3experiment.

FIGS. 41A-B shows VCC data (A) before lyophilization andpost-lyophilization immediately after lyophilization and after storageat 30° C. for 24, 48, or 72 hours, and residual moisture (B) in the WP6experiment.

FIGS. 42A-B show lyophilization cakes (A) and reconstitution times (B)post-lyophilization immediately after lyophilization and after storageat 30° C. for 24, 48, or 72 hours in the WP7 experiment.

FIG. 43 shows VCC data before lyophilization and post-lyophilizationimmediately after lyophilization and after storage at 30° C. for 24hours, 72 hours, and 7 days in the WP7 experiment.

FIGS. 44A-B show VCC data (A) and percent live cells (B) vs. storagetime at 30° C. in the WP7 experiment.

FIGS. 45A-B show CFU/mL (A) and percent viable cells (B) vs. time in theWP7 experiment.

FIGS. 46A-B show VCC data before lyophilization and post-lyophilizationimmediately after lyophilization and after storage at 30° C. for 24 and72 hours in the WP7 experiment.

FIGS. 47A-B show VCC data before lyophilization and post-lyophilizationimmediately after lyophilization and after storage at 30° C. for 24 and72 hours in the WP7 experiment.

FIG. 48 shows a scatterplot of VCC and RM for ADXS11-001 Pilot Batch.

FIG. 49 shows a scatterplot of VCC for Lot #5329PD-17-01 (ADXS11-001Pilot Batch).

FIG. 50 show a scatterplot of % live for Lot #5329PD-17-01 (ADXS11-001Pilot Batch).

FIG. 51 show a scatterplot of pH for Lot #5329PD-17-01 (ADXS11-001 PilotBatch).

FIG. 52 shows a scatterplot of VCC for Lot #5329PD-17-01 stored at 30°C. (ADXS11-001 Pilot Batch).

FIG. 53 shows a scatterplot of % live for Lot #5329PD-17-01 stored at30° C. (ADXS11-001 Pilot Batch).

FIG. 54 shows a chart illustrating implantation and dosing schedule(ADXS11-001 Pilot Batch).

FIG. 55 shows graphs illustrating both lyophilized AXAL and clinicalAXAL inhibit tumor growth in the TC-1 tumor model at different doses.

FIG. 56 shows graphs illustrating both lyophilized AXAL and clinicalAXAL prolong animal survival in the TC-1 tumor model at different doses.

FIG. 57 shows a graph illustrating reconstitution time of WP7 cycle 3compared to cycle 1 and cycle 2.

FIGS. 58A-B show graphs illustrating MFI analysis at Tliq, Tlyo, andafter storage for 24 and 72 hours at 30° C. of A0085 (A) and A1300 (B).

FIGS. 58C-D show graphs illustrating MFI analysis at Tliq, Tlyo, andafter storage for 24 and 72 hours at 30° C. of B0085 (C) and B1300 (D).

FIGS. 59A-B show graphs illustrating RRM results for A. A0085 (200 folddilution) and B. A1300 (5,000-fold dilution).

FIGS. 59C-D show graphs illustrating RRM results for C. B0085 (200 folddilution) and D. B1300 (5,000-fold dilution).

FIGS. 60A-B show graphs illustrating negatively buoyant particledistribution for A0085 (200 fold dilution) (A) and A1300 (5,000-folddilution) (B).

FIGS. 60C-D show graphs illustrating negatively buoyant particledistribution for B0085 (200 fold dilution) (C) and B1300 (5,000-folddilution) (D).

FIG. 61 shows a graph illustrating Karl-Fischer titration analysis offive vials at Tlyo of A0085, A1300, B0085, and B1300.

FIG. 62 shows a graph illustrating results of VCC assay performed atTliq, Tlyo and after storage for 24 h, 48 h, and 72 h at 30° C. Frontvials were stored at −20° C. for 7 days before analysis.

FIG. 63A shows a scatterplot illustrating VCC after WP7 Cycle 3 onaccelerated stability.

FIG. 63B shows a scatterplot illustrating % live after WP7 Cycle 3 onaccelerated stability.

FIG. 64 shows a scatterplot illustrating impact on VCC of repeatedfreeze/thaw cycles on BDS (1 L Fill/1 L LDPE Bag) at varioustemperatures and VCC levels.

FIG. 65 shows a scatterplot illustrating impact on % live of repeatedfreeze/thaw cycles on BDS (1 L Fill/1 L LDPE Bag) at varioustemperatures and VCC levels.

DEFINITIONS

The terms “protein,” “polypeptide,” and “peptide,” used interchangeablyherein, refer to polymeric forms of amino acids of any length, includingcoded and non-coded amino acids and chemically or biochemically modifiedor derivatized amino acids. The terms include polymers that have beenmodified, such as polypeptides having modified peptide backbones.

Proteins are said to have an “N-terminus” and a “C-terminus.” The term“N-terminus” relates to the start of a protein or polypeptide,terminated by an amino acid with a free amine group (—NH2). The term“C-terminus” relates to the end of an amino acid chain (protein orpolypeptide), terminated by a free carboxyl group (—COOH).

The term “fusion protein” refers to a protein comprising two or morepeptides linked together by peptide bonds or other chemical bonds. Thepeptides can be linked together directly by a peptide or other chemicalbond. For example, a chimeric molecule can be recombinantly expressed asa single-chain fusion protein. Alternatively, the peptides can be linkedtogether by a “linker” such as one or more amino acids or anothersuitable linker between the two or more peptides.

The terms “nucleic acid” and “polynucleotide,” used interchangeablyherein, refer to polymeric forms of nucleotides of any length, includingribonucleotides, deoxyribonucleotides, or analogs or modified versionsthereof. They include single-, double-, and multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purinebases, pyrimidine bases, or other natural, chemically modified,biochemically modified, non-natural, or derivatized nucleotide bases.

Nucleic acids are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. An end of an oligonucleotide is referred to as the “5′ end” ifits 5′ phosphate is not linked to the 3′ oxygen of a mononucleotidepentose ring. An end of an oligonucleotide is referred to as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of anothermononucleotide pentose ring. A nucleic acid sequence, even if internalto a larger oligonucleotide, also may be said to have 5′ and 3′ ends. Ineither a linear or circular DNA molecule, discrete elements are referredto as being “upstream” or 5′ of the “downstream” or 3′ elements.

“Codon optimization” refers to a process of modifying a nucleic acidsequence for enhanced expression in particular host cells by replacingat least one codon of the native sequence with a codon that is morefrequently or most frequently used in the genes of the host cell whilemaintaining the native amino acid sequence. For example, apolynucleotide encoding a fusion polypeptide can be modified tosubstitute codons having a higher frequency of usage in a given Listeriacell or any other host cell as compared to the naturally occurringnucleic acid sequence. Codon usage tables are readily available, forexample, at the “Codon Usage Database.” The optimal codons utilized byL. monocytogenes for each amino acid are shown US 2007/0207170, hereinincorporated by reference in its entirety for all purposes. These tablescan be adapted in a number of ways. See Nakamura et al. (2000) NucleicAcids Research 28:292, herein incorporated by reference in its entiretyfor all purposes. Computer algorithms for codon optimization of aparticular sequence for expression in a particular host are alsoavailable (see, e.g., Gene Forge).

The term “plasmid” or “vector” includes any known delivery vectorincluding a bacterial delivery vector, a viral vector delivery vector, apeptide immunotherapy delivery vector, a DNA immunotherapy deliveryvector, an episomal plasmid, an integrative plasmid, or a phage vector.The term “vector” refers to a construct which is capable of delivering,and, optionally, expressing, one or more fusion polypeptides in a hostcell.

The term “episomal plasmid” or “extrachromosomal plasmid” refers to anucleic acid vector that is physically separate from chromosomal DNA(i.e., episomal or extrachromosomal and does not integrated into a hostcell's genome) and replicates independently of chromosomal DNA. Aplasmid may be linear or circular, and it may be single-stranded ordouble-stranded. Episomal plasmids may optionally persist in multiplecopies in a host cell's cytoplasm (e.g., Listeria), resulting inamplification of any genes of interest within the episomal plasmid.

The term “genomically integrated” refers to a nucleic acid that has beenintroduced into a cell such that the nucleotide sequence integrates intothe genome of the cell and is capable of being inherited by progenythereof. Any protocol may be used for the stable incorporation of anucleic acid into the genome of a cell.

The term “stably maintained” refers to maintenance of a nucleic acidmolecule or plasmid in the absence of selection (e.g., antibioticselection) for at least 10 generations without detectable loss. Forexample, the period can be at least 15 generations, 20 generations, atleast 25 generations, at least 30 generations, at least 40 generations,at least 50 generations, at least 60 generations, at least 80generations, at least 100 generations, at least 150 generations, atleast 200 generations, at least 300 generations, or at least 500generations. Stably maintained can refer to a nucleic acid molecule orplasmid being maintained stably in cells in vitro (e.g., in culture),being maintained stably in vivo, or both.

An “open reading frame” or “ORF” is a portion of a DNA which contains asequence of bases that could potentially encode a protein. As anexample, an ORF can be located between the start-code sequence(initiation codon) and the stop-codon sequence (termination codon) of agene.

A “promoter” is a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particularpolynucleotide sequence. A promoter may additionally comprise otherregions which influence the transcription initiation rate. The promotersequences disclosed herein modulate transcription of an operably linkedpolynucleotide. A promoter can be active in one or more of the celltypes disclosed herein (e.g., a eukaryotic cell, a non-human mammaliancell, a human cell, a rodent cell, a pluripotent cell, a one-cell stageembryo, a differentiated cell, or a combination thereof). A promoter canbe, for example, a constitutively active promoter, a conditionalpromoter, an inducible promoter, a temporally restricted promoter (e.g.,a developmentally regulated promoter), or a spatially restrictedpromoter (e.g., a cell-specific or tissue-specific promoter). Examplesof promoters can be found, for example, in WO 2013/176772, hereinincorporated by reference in its entirety.

“Operable linkage” or being “operably linked” refers to thejuxtaposition of two or more components (e.g., a promoter and anothersequence element) such that both components function normally and allowthe possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Forexample, a promoter can be operably linked to a coding sequence if thepromoter controls the level of transcription of the coding sequence inresponse to the presence or absence of one or more transcriptionalregulatory factors. Operable linkage can include such sequences beingcontiguous with each other or acting in trans (e.g., a regulatorysequence can act at a distance to control transcription of the codingsequence).

“Sequence identity” or “identity” in the context of two polynucleotidesor polypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well-known.Typically, this involves scoring a conservative substitution as apartial rather than a full mismatch, thereby increasing the percentagesequence identity. Thus, for example, where an identical amino acid isgiven a score of 1 and a non-conservative substitution is given a scoreof zero, a conservative substitution is given a score between zeroand 1. The scoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

“Percentage of sequence identity” refers to the value determined bycomparing two optimally aligned sequences (greatest number of perfectlymatched residues) over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity. Unless otherwise specified(e.g., the shorter sequence includes a linked heterologous sequence),the comparison window is the full length of the shorter of the twosequences being compared.

Unless otherwise stated, sequence identity/similarity values refer tothe value obtained using GAP Version 10 using the following parameters:% identity and % similarity for a nucleotide sequence using GAP Weightof 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; %identity and % similarity for an amino acid sequence using GAP Weight of8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or anyequivalent program thereof. “Equivalent program” includes any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The term “conservative amino acid substitution” refers to thesubstitution of an amino acid that is normally present in the sequencewith a different amino acid of similar size, charge, or polarity.Examples of conservative substitutions include the substitution of anon-polar (hydrophobic) residue such as isoleucine, valine, or leucinefor another non-polar residue. Likewise, examples of conservativesubstitutions include the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, or between glycine and serine. Additionally,the substitution of a basic residue such as lysine, arginine, orhistidine for another, or the substitution of one acidic residue such asaspartic acid or glutamic acid for another acidic residue are additionalexamples of conservative substitutions. Examples of non-conservativesubstitutions include the substitution of a non-polar (hydrophobic)amino acid residue such as isoleucine, valine, leucine, alanine, ormethionine for a polar (hydrophilic) residue such as cysteine,glutamine, glutamic acid or lysine and/or a polar residue for anon-polar residue. Typical amino acid categorizations are summarized inTable 1 below.

TABLE 1 Amino Acid Categorizations. Alanine Ala A Nonpolar Neutral 1.8Arginine Arg R Polar Positive −4.5 Asparagine Asn N Polar Neutral −3.5Aspartic acid Asp D Polar Negative −3.5 Cysteine Cys C Nonpolar Neutral2.5 Glutamic acid Glu E Polar Negative −3.5 Glutamine Gln Q PolarNeutral −3.5 Glycine Gly G Nonpolar Neutral −0.4 Histidine His H PolarPositive −3.2 Isoleucine Ile I Nonpolar Neutral 4.5 Leucine Leu LNonpolar Neutral 3.8 Lysine Lys K Polar Positive −3.9 Methionine Met MNonpolar Neutral 1.9 Phenylalanine Phe F Nonpolar Neutral 2.8 ProlinePro P Nonpolar Neutral −1.6 Serine Ser S Polar Neutral −0.8 ThreonineThr T Polar Neutral −0.7 Tryptophan Trp W Nonpolar Neutral −0.9 TyrosineTyr Y Polar Neutral −1.3 Valine Val V Nonpolar Neutral 4.2

A “homologous” sequence (e.g., nucleic acid sequence) refers to asequence that is either identical or substantially similar to a knownreference sequence, such that it is, for example, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the knownreference sequence.

The term “wild type” refers to entities having a structure and/oractivity as found in a normal (as contrasted with mutant, diseased,altered, or so forth) state or context. Wild type gene and polypeptidesoften exist in multiple different forms (e.g., alleles).

The term “isolated” with respect to proteins and nucleic acid refers toproteins and nucleic acids that are relatively purified with respect toother bacterial, viral or cellular components that may normally bepresent in situ, up to and including a substantially pure preparation ofthe protein and the polynucleotide. The term “isolated” also includesproteins and nucleic acids that have no naturally occurring counterpart,have been chemically synthesized and are thus substantiallyuncontaminated by other proteins or nucleic acids, or has been separatedor purified from most other cellular components with which they arenaturally accompanied (e.g., other cellular proteins, polynucleotides,or cellular components).

“Exogenous” or “heterologous” molecules or sequences are molecules orsequences that are not normally expressed in a cell or are not normallypresent in a cell in that form. Normal presence includes presence withrespect to the particular developmental stage and environmentalconditions of the cell. An exogenous or heterologous molecule orsequence, for example, can include a mutated version of a correspondingendogenous sequence within the cell or can include a sequencecorresponding to an endogenous sequence within the cell but in adifferent form (i.e., not within a chromosome). An exogenous orheterologous molecule or sequence in a particular cell can also be amolecule or sequence derived from a different species than a referencespecies of the cell or from a different organism within the samespecies. For example, in the case of a Listeria strain expressing aheterologous polypeptide, the heterologous polypeptide could be apolypeptide that is not native or endogenous to the Listeria strain,that is not normally expressed by the Listeria strain, from a sourceother than the Listeria strain, derived from a different organism withinthe same species.

In contrast, “endogenous” molecules or sequences or “native” moleculesor sequences are molecules or sequences that are normally present inthat form in a particular cell at a particular developmental stage underparticular environmental conditions.

The term “variant” refers to an amino acid or nucleic acid sequence (oran organism or tissue) that is different from the majority of thepopulation but is still sufficiently similar to the common mode to beconsidered to be one of them (e.g., splice variants).

The term “isoform” refers to a version of a molecule (e.g., a protein)with only slight differences compared to another isoform, or version(e.g., of the same protein). For example, protein isoforms may beproduced from different but related genes, they may arise from the samegene by alternative splicing, or they may arise from single nucleotidepolymorphisms.

The term “fragment” when referring to a protein means a protein that isshorter or has fewer amino acids than the full length protein. The term“fragment” when referring to a nucleic acid means a nucleic acid that isshorter or has fewer nucleotides than the full length nucleic acid. Afragment can be, for example, an N-terminal fragment (i.e., removal of aportion of the C-terminal end of the protein), a C-terminal fragment(i.e., removal of a portion of the N-terminal end of the protein), or aninternal fragment. A fragment can also be, for example, a functionalfragment or an immunogenic fragment.

The term “analog” when referring to a protein means a protein thatdiffers from a naturally occurring protein by conservative amino aciddifferences, by modifications which do not affect amino acid sequence,or by both.

The term “functional” refers to the innate ability of a protein ornucleic acid (or a fragment, isoform, or variant thereof) to exhibit abiological activity or function. Such biological activities or functionscan include, for example, the ability to elicit an immune response whenadministered to a subject. Such biological activities or functions canalso include, for example, binding to an interaction partner. In thecase of functional fragments, isoforms, or variants, these biologicalfunctions may in fact be changed (e.g., with respect to theirspecificity or selectivity), but with retention of the basic biologicalfunction.

The terms “immunogenicity” or “immunogenic” refer to the innate abilityof a molecule (e.g., a protein, a nucleic acid, an antigen, or anorganism) to elicit an immune response in a subject when administered tothe subject. Immunogenicity can be measured, for example, by a greaternumber of antibodies to the molecule, a greater diversity of antibodiesto the molecule, a greater number of T-cells specific for the molecule,a greater cytotoxic or helper T-cell response to the molecule, and thelike.

The term “antigen” is used herein to refer to a substance that, whenplaced in contact with a subject or organism (e.g., when present in orwhen detected by the subject or organism), results in a detectableimmune response from the subject or organism. An antigen may be, forexample, a lipid, a protein, a carbohydrate, a nucleic acid, orcombinations and variations thereof. For example, an “antigenic peptide”refers to a peptide that leads to the mounting of an immune response ina subject or organism when present in or detected by the subject ororganism. For example, such an “antigenic peptide” may encompassproteins that are loaded onto and presented on MHC class I and/or classII molecules on a host cell's surface and can be recognized or detectedby an immune cell of the host, thereby leading to the mounting of animmune response against the protein. Such an immune response may alsoextend to other cells within the host, such as diseased cells (e.g.,tumor or cancer cells) that express the same protein.

The term “epitope” refers to a site on an antigen that is recognized bythe immune system (e.g., to which an antibody binds). An epitope can beformed from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of one or more proteins. Epitopes formedfrom contiguous amino acids (also known as linear epitopes) aretypically retained on exposure to denaturing solvents whereas epitopesformed by tertiary folding (also known as conformational epitopes) aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, and more usually, at least 5 or 8-10amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996), herein incorporated by reference in itsentirety for all purposes.

The term “mutation” refers to the any change of the structure of a geneor a protein. For example, a mutation can result from a deletion, aninsertion, a substitution, or a rearrangement of chromosome or aprotein. An “insertion” changes the number of nucleotides in a gene orthe number of amino acids in a protein by adding one or more additionalnucleotides or amino acids. A “deletion” changes the number ofnucleotides in a gene or the number of amino acids in a protein byreducing one or more additional nucleotides or amino acids.

A “frameshift” mutation in DNA occurs when the addition or loss ofnucleotides changes a gene's reading frame. A reading frame consists ofgroups of 3 bases that each code for one amino acid. A frameshiftmutation shifts the grouping of these bases and changes the code foramino acids. The resulting protein is usually nonfunctional. Insertionsand deletions can each be frameshift mutations.

A “missense” mutation or substitution refers to a change in one aminoacid of a protein or a point mutation in a single nucleotide resultingin a change in an encoded amino acid. A point mutation in a singlenucleotide that results in a change in one amino acid is a“nonsynonymous” substitution in the DNA sequence. Nonsynonymoussubstitutions can also result in a “nonsense” mutation in which a codonis changed to a premature stop codon that results in truncation of theresulting protein. In contrast, a “synonymous” mutation in a DNA is onethat does not alter the amino acid sequence of a protein (due to codondegeneracy).

The term “somatic mutation” includes genetic alterations acquired by acell other than a germ cell (e.g., sperm or egg). Such mutations can bepassed on to progeny of the mutated cell in the course of cell divisionbut are not inheritable. In contrast, a germinal mutation occurs in thegerm line and can be passed on to the next generation of offspring.

The term “in vitro” refers to artificial environments and to processesor reactions that occur within an artificial environment (e.g., a testtube).

The term “in vivo” refers to natural environments (e.g., a cell ororganism or body) and to processes or reactions that occur within anatural environment.

The term “frozen state glass transition temperature” (Tg′) refers to thefollowing. When heated, solutions of sugar glasses undergo asecond-order transition from a rigid state to a viscoelastic rubberystate. The temperature at which the vitreous transformation occurs isthe glass-transition temperature in the frozen state.

The term “solid state glass transition temperature” (Tg) refers to thefollowing. Similar to Tg′, this is the temperature at which thefreeze-dried glassy solid transforms to a viscoelastic rubbery state.

The term “collapse temperature” (Tc) refers to the maximum temperaturethat the product can withstand during primary drying without losing itsphysical structure.

The term “drug substance” (DS) refers to an active ingredient. It refersto any component of a drug product intended to furnish pharmacologicalactivity or other direct effect in the diagnosis, cure, mitigation,treatment, or prevention of disease, or to affect the structure or anyfunction of the body of humans or other animals. Active ingredientsinclude those components of the product that may undergo chemical changeduring the manufacture of the drug product and be present in the drugproduct in a modified form intended to furnish the specified activity oreffect. For example, Lm (e.g., ADXS-HPV or ADXS-HER2) is considered adrug substance.

The term “bulk drug substance” (BDS) refers to any substance that isrepresented for use in a drug and that, when used in the manufacturing,processing, or packaging of a drug, becomes an active ingredient or afinished dosage form of the drug, but the term does not includeintermediates used in the synthesis of such substances.

The term “drug product” (DP) refers to a finished dosage form, forexample, a tablet, capsule or solution that contains an activepharmaceutical ingredient, generally, but not necessarily, inassociation with inactive ingredients. For example, lyophilized Lm(e.g., ADXS-HPV or ADXS-HER2) is considered a drug product.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” a protein maycontain the protein alone or in combination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (e.g., SEM) of astated value or variations±0.5%, 1%, 5%, or 10% from a specified value.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “an antigen” or “at least one antigen” can include a pluralityof antigens, including mixtures thereof.

Statistically significant means p≤0.05.

DETAILED DESCRIPTION

I. Overview

Disclosed herein are compositions and methods directed to a stablelyophilized pharmaceutical formulation prepared by lyophilizing anaqueous formulation comprising a bacteria or Listeria strain, such asListeria monocytogenes. In some embodiments, the lyophilized formulationis stable at 4° C. or −20° C. for at least 6 months, at least 1 year, orat least 2 years. In some embodiments, the lyophilized formulation issuitable for parenteral administration such as intravenous injection.

The frozen liquid formulations currently used for therapeuticscomprising, for example, L. monocytogenes, are stored and shipped at−80° C. The low temperature presents supply chain challenges with bothshipping and storage of the material at clinical sites, particularly incountries in South America and Africa. Therefore, it is desirable tohave a refrigerated or −20° C. supply chain. Through optimization of themanufacturing process as described herein, it is possible to generate astable drug product that is able to be maintained at highertemperatures. Counterintuitively, the working examples described hereinshow that higher residual moistures (e.g., higher than normal targetedresidual moisture levels such as about 2.5%, 3.0%, or 3.5% in oneembodiment) improved the stability of the lyophilized product.Similarly, counterintuitively, higher shelf temperatures during theprimary drying step (e.g., about −17° C. to about −19° C. or about −18°C. in one embodiment, which is well above the Tg) improved the stabilityof the lyophilized product. In addition, preconditioning of the cellsprior to lyophilization through heat shock improved the stability of thelyophilized product. In addition, use of a higher concentration ofviable bacteria (viable cell count, or VCC) results in an improvement inthe stability of the lyophilized drug product relative to lower VCC. Inaddition, counterintuitively, thawing of frozen drug substance at about37° C. prior to lyophilization improved stability of the lyophilizeddrug product relative to thawing at room temperature or 2-8° C. Theseprocess enhancements improve stability at higher temperatures comparedto a liquid frozen formulation allowing for a higher temperature supplychain. This allows for a more manageable supply chain and distributionto countries were −80° C. storage is not feasible.

The process of inducing a stress response within the L. monocytogenescells through a temperature shift prior to lyophilization in addition tooptimization of the residual moisture in the lyophilized cake (e.g.,using higher than normal targeted residual moisture levels such as about3.5% in one embodiment, which can be achieved by altering the secondarydrying temperature and optionally the secondary drying time) improvestability of the lyophilized drug product at higher temperatures.Similarly, use of formulations comprising a phosphate buffer and lowerthan normal levels of sucrose (e.g., about 2.5% w/v sucrose in oneembodiment) and using high primary drying step temperatures (e.g., about−18° C. in one embodiment) improve stability of the lyophilized drugproduct at higher temperatures, including −20° C., 2-8° C., and evenroom temperature (about 20° C. to about 25° C., or about 20° C., about23° C., or about 25° C.). These process enhancements improve stabilityat higher temperatures compared to a liquid frozen formulation allowingfor a higher temperature supply chain. This allows for a more manageablesupply chain and distribution to countries were −80° C. storage is notfeasible.

II. Lyophilization of Bacteria or Listeria

Lyophilization can be divided in three steps: freezing, primary drying,and secondary drying. As water freezes in the first step, the dissolvedcomponents in the formulation remain in the residual liquid (thefreeze-concentrate). At the point of maximal ice formation, thefreeze-concentrate solidifies between the ice crystals that make up thelattice. Under appropriate lyophilization conditions, the ice is removedby sublimation during primary drying, leaving the remainingfreeze-concentrate in the same physical and chemical structure as whenthe ice was present. Residual water in the freeze-concentrate is removedin the secondary drying step.

Lyophilization involves manipulating the temperature and pressure of thesolution so that the phase of the solvent can move directly from thefrozen state to the gaseous state without moving through the liquidphase/state. This is achieved by cooling the solution and lowering thepressure to below the triple point of water. This allows for the removalof the solvent from the product without subjecting the product tointense heat. During the freezing stage, the formulation is cooled. Purecrystalline ice forms from the liquid, thereby resulting in a freezeconcentration of the remainder of the liquid to a more viscous statethat inhibits further crystallization. Ultimately, this highlyconcentrated and viscous solution solidifies, yielding an amorphous,crystalline, or combined amorphous-crystalline phase. During the primarydrying stage, the ice formed during freezing is removed by sublimationat sub-ambient temperatures under vacuum. Throughout this stage, theproduct is maintained in the solid state below the collapse temperatureof the product in order to dry the product with retention of thestructure established in the freezing step. The collapse temperature isthe glass transition temperature (Tg′) in the case of amorphous productsor the eutectic temperature (Te) for crystalline products. During thesecondary drying stage, the relatively small amount of bound waterremaining in the matrix is removed by desorption. During this stage, thetemperature of the shelf and product are increased to promote adequatedesorption rates and achieve the desired residual moisture.

The target profile for a lyophilized drug product is one that produces awell-defined cake at a target residual moisture that is stable at either2-8° C. or −20° C. and retains the same potency and biological activityas the liquid-frozen formulation. Protection strategies that may enhancebacterial viability during freeze drying include, for example, addingexcipients to the drying medium, controlling the process parameters,pre-stressing the bacterial sample prior to freeze drying, and changingthe fermentation conditions of the bacteria. However, the efficiency ofthese strategies is strain-dependent, because the intrinsic tolerance tothe drying process varies also from strain to strain. Even in highlyrelated bacteria strains, one strain may be much more resistant to thefreeze-drying process than the other. This strain dependency makes itdifficult to draw general conclusions and guidelines.

Provided herein are methods for producing a lyophilized compositioncomprising a bacteria or Listeria strain. Such methods can compriseproviding a composition comprising a bacteria or Listeria strain in aformulation comprising a buffer, cooling the composition in a freezingstep, exposing the cooled composition to a vacuum and a first increasedtemperature in a primary drying step, and exposing the composition fromthe primary drying step to a vacuum and a second increased temperaturein a secondary drying step, whereby the lyophilized composition isproduced.

In some such methods, the bacteria or Listeria strain used in thecomposition is a frozen Listeria strain that is thawed prior to thefreezing step. Examples of such preconditioning steps are described inmore detail elsewhere herein. In a specific example, the frozen bacteriaor Listeria strain can be thawed at a temperature of about 2° C. toabout 37° C., about 20° C. to about 37° C., about 23° C. to about 37°C., about 25° C. to about 37° C., about 32° C. to about 37° C., or about37° C. Optionally, the thawing is for no more than about 8 hours.Optionally, the thawed bacteria or Listeria strain is held attemperature of between about 2° C. and about 8° C. for no more thanabout 24 hours. In a specific example, the concentration of the bacteriaor Listeria strain being thawed can be between about 1×10E9 and about1×10E10 colony forming units (CFU) per milliliter.

In some such methods, the formulation comprises a buffer and sucrose.For example, the formulation buffer can comprise about 1% to about 5%w/v sucrose, about 2% to about 3% w/v sucrose, or about 2.5% w/vsucrose. Optionally, the formulation does not comprise other excipientssuch as trehalose, monosodium glutamate (MSG), or recombinant humanserum albumin (rHSA).

In some such methods, the formulation comprises about 1×10E9 to about1×10E10 colony forming units (CFU) of bacteria or Listeria permilliliter.

In some such methods, the holding temperature in the primary drying stepis between about −10° C. and about −30° C., between about −12° C. andabout −22° C., between about −17° C. and about −19° C., or about −18° C.

In some such methods, the residual moisture in the lyophilizedcomposition is at least about 2.5%, at least about 3%, or at least about3.5%. In some such methods, the residual moisture is between about 1%and about 5% or between about 2% and about 4%.

In some such methods, the lyophilized composition shows at least about60%, 70%, 80%, or 90% viability after storage at between about −20° C.and about 4° C. or after storage at about −20° C. or about 4° C. forabout 6 months, 12 months, 18 months, or 24 months.

Some such methods comprise: (a) providing a composition comprising abacteria or Listeria strain in a formulation comprising a buffer andsucrose; (b) cooling the composition provided in step (a) at a holdingtemperature between about −32° C. and about −80° C. in a freezing step;(c) exposing the composition produced by step (b) to a vacuum at aholding temperature between about −10° C. and about −30° C. in a primarydrying step; and (d) exposing the composition produced by step (c) to avacuum at a holding temperature between about −5° C. and about 25° C. ina secondary drying step.

Additional embodiments for the preconditioning of cells, theformulations, the freezing step, the primary drying step, the secondarydrying step, and the lyophilized product are provided below.

A. Pre-Conditioning of Bacteria or Listeria

A culture of a bacteria or Listeria strain that is used in alyophilization method disclosed herein can be from a frozen stock, froma starter culture, or from a colony (e.g., freshly cultured bacteria orListeria).

Methods are provided herein for preparing a frozen bacteria or Listeriastrain for lyophilization, comprising thawing the frozen bacteria orListeria strain. If the bacteria or Listeria strain is from a frozenstock, it can be thawed by any means. Temperature and the time forthawing can impact stability. Identifying appropriate conditions forthawing frozen drug substance allows freezing and holding of the drugsubstance prior to lyophilization. Ensuring high-quality healthy cellscoming out of thaw ensures that the resulting lyophilized drug productis also of sufficient quality. In one example, it can be thawed at about−4° C., about 2-8° C., or about 4° C. and incubated, for example, forabout 0.5, 1, 2, 3, 4, or more hours. In another example, it can bethawed at about 37° C. and incubated, for example, for about 0.5, 1, 2,3, 4, or more hours.

In one example, the frozen bacteria or Listeria strain can be thawed atemperature between about 4° C. and about 37° C., about 10° C. and about37° C., about 15° C. and about 37° C., about 20° C. and about 37° C.,about 23° C. and about 37° C., about 25° C. and about 37° C., about 25°C. and about 37° C., about 30° C. and about 37° C., about 32° C. andabout 37° C., about 32° C. and about 42° C., about 34° C. and about 40°C., about 35° C. and about 39° C., about 36° C. and 38° C., or about 37°C.

The frozen bacteria or Listeria strain can be thawed, for example, forabout 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours, for between about 0.5 andabout 8 hours, between about 1 and about 8 hours, between about 2 andabout 8 hours, between about 3 and about 8 hours, between about 4 andabout 8 hours, between about 5 and about 8 hours, between about 6 andabout 8 hours, or between about 7 and about 8 hours. Alternatively, thefrozen bacteria or Listeria strain can be thawed, for example, for nomore than about 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 hours.

The frozen bacteria or Listeria strain being thawed can be in a bacteriaor Listeria lyophilization formulation or can be thawed in a bacteria orListeria lyophilization formulation. Such bacteria or Listerialyophilization formulations are disclosed in more detail elsewhereherein.

The frozen bacteria or Listeria strain can be held at a temperatureafter thawing. For example, the frozen bacteria or Listeria strain canbe held at a temperature of between about 2° C. to about 8° C. afterthawing. The frozen bacteria or Listeria strain can be held, forexample, for about 0.5 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or between about 0.5 andabout 24 hours, between about 1 and about 24 hours, between about 2 andabout 24 hours, between about 5 and about 24 hours, between about 10 andabout 24 hours, between about 12 and about 24 hours. Alternatively, thefrozen bacteria or Listeria strain can be held, for example, for no morethan about 0.5 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or 24 hours. In a specific example, thefrozen bacteria or Listeria strain is thawed at a temperature of about37° C. for no more than about 8 hours and is held at temperature ofbetween about 2° C. and about 8° C. for no more than about 24 hours.

The concentration of the bacteria or Listeria strain being thawed can beany suitable concentration. For example, the concentration can bebetween about 1×10E9 and about 1×10E10 colony forming units (CFU) permilliliter.

The culture used for lyophilization can be at any growth phase. Theculture can be, for example, at mid-log growth phase, at approximatelymid-log growth phase, or at another growth phase.

The nutrient medium utilized for growing a culture of a bacteria orListeria strain can be any suitable nutrient medium. Examples ofsuitable media include, for example, Luria broth (LB; Luria-Bertanibroth); Terrific Broth (TB); a modified, animal-product-free TerrificBroth; or a defined medium. The bacteria or Listeria strain can becultured by any known means of growing bacteria. For example, the stepof growing can be performed with a shake flask (such as a baffled shakeflask), a batch fermenter, a stirred tank or flask, an airliftfermenter, a fed batch, a continuous cell reactor, an immobilized cellreactor, or any other means of growing bacteria.

Optionally, a constant pH is maintained during growth of the culture(e.g. in a batch fermenter). For example, the pH can be maintained atabout 6.0, at about 6.5, at about 7.0, at about 7.5, or about 8.0.Likewise, the pH can be, for example, from about 6.5 to about 7.5, fromabout 6.0 to about 8.0, from about 6.0 to about 7.0, from about 6.0 toabout 7.0, or from about 6.5 to about 7.5. Alternatively, immediatelyafter harvesting the cells from the bioreactor, the pH can be dropped bythe addition of acid to induce a stress response, which can activate aset of genes that may better prepare the cells for lyophilization.

Optionally, a constant temperature can be maintained during growth ofthe culture. For example, the temperature can be maintained at about 37°C. Alternatively, the temperature can be maintained at about 25° C.,about 27° C., about 28° C., about 30° C., about 32° C., about 34° C.,about 35° C., about 36° C., about 38° C., or about 39° C. Alternatively,immediately after harvesting the cells from the bioreactor, thetemperature can be dropped by placing the cells in an ice bath (e.g.,about 0° C. or about 4° C.) to induce a stress response, which canactivate a set of genes that may better prepare the cells forlyophilization.

Optionally, a constant dissolved oxygen concentration can be maintainedduring growth of the culture. For example, the dissolved oxygenconcentration can be maintained at 20% of saturation, 15% of saturation,16% of saturation, 18% of saturation, 22% of saturation, 25% ofsaturation, 30% of saturation, 35% of saturation, 40% of saturation, 45%of saturation, 50% of saturation, 55% of saturation, 60% of saturation,65% of saturation, 70% of saturation, 75% of saturation, 80% ofsaturation, 85% of saturation, 90% of saturation, 95% of saturation,100% of saturation, or near 100% of saturation.

The bacteria strain or Listeria strain can optionally be passagedthrough an animal host prior to lyophilization. Such passaging canmaximize efficacy of the Listeria strain as a vaccine vector, canstabilize the immunogenicity of the Listeria strain, can stabilize thevirulence of the Listeria strain, can increase the immunogenicity of theListeria strain, can increase the virulence of the Listeria strain, canremove unstable sub-strains of the Listeria strain, or can reduce theprevalence of unstable sub-strains of the Listeria strain. Methods forpassaging a Listeria strain through an animal host are well-known andare described, for example, in US 2006/0233835, herein incorporated byreference in its entirety for all purposes.

B. Bacteria or Listeria Lyophilization Formulations

Prior to the lyophilization, the bacteria or Listeria strain can beprovided in a suspension (formulation) comprising a buffer and anexcipient. The design of a lyophilized formulation can depend on therequirements of the active pharmaceutical ingredient and the intendedroute of administration. A formulation may consist of a buffer and oneor more excipients that perform one or more functions. Such excipientscan be, for example, pH adjusters, bulking agents (e.g., sucrose,mannitol, maltose, trehalose, dextrose, and lactose), stabilizers suchas cryoprotectants (e.g., PEG) and lyoprotectants (e.g., disaccharides),or tonicity modifiers (e.g., NaCl, mannitol, sucrose, glycine, andglycerol).

The buffer can be any suitable buffer. Buffers can stabilize pH in theformulation. For example, the buffer can be a phosphate buffer, a Trisbuffer, a histidine buffer, a citrate buffer, or a MOPS(3-(N-morpholino)propanesulfonic acid) buffer. In a specific example,the buffer is a phosphate buffer. Phosphate buffers are often avoided inthe development of lyophilized formulations because phosphate buffers,such as sodium phosphate, can undergo drastic pH changes duringfreezing. Because of this, low concentrations of buffers that undergominimal pH changes during freezing, such as Tris, citrate, and histidinebuffers are often used. However, as shown elsewhere herein, suitableviability levels of Listeria monocytogenes are achieved using phosphatebuffers. In some such buffers, the concentration of KH₂PO₄ (anhydrous)is between about 0.1-0.3 g/L, 0.12-0.28 g/L, 0.14-0.26 g/L, 0.16-0.24g/L, 0.18-0.22 g/L, 0.19-0.21 g/L, or 0.2 g/L. In some such buffers, theconcentration of Na₂HPO₄ (anhydrous) is between about 1.0-1.3 g/L,1.02-1.28 g/L, 1.04-1.26 g/L, 1.06-1.24 g/L, 1.08-1.22 g/L, 1.1-1.2 g/L,1.12-1.18 g/L, 1.14-1.16 g/L, or 1.15 g/L. Some such buffers are about5-20, 6-18, 7-16, 8-14, 9-12, 9-11, or 10 mM. Some such buffers have apH of about 6.8-7.6, 6.9-7.5, 7.0-7.4, 7.1-7.3, or 7.2. As one example,the phosphate buffer can have between about 0.19-0.21 g/L (e.g., 0.2g/L) of KH₂PO₄ (anhydrous), between about 1.14-1.16 g/L (e.g., 1.15 g/L)of Na₂HPO₄ (anhydrous), and can have a pH of about 7.1-7.3 (e.g., 7.2).

Excipients such as cryoprotectants and lyoprotectants can be added tothe formulation to protect the bacteria or Listeria strain during thelyophilization process. Cryoprotectants are water-soluble chemicals thatlower the melting point of water. As ice crystals are formed, bacterialcells are compressed in the unfrozen fraction. Adding cryoprotectantscan enlarge the unfrozen section, giving more space to the bacterialcells, which can lead to less cellular damage by mechanical stress orosmotic stress. Lyoprotectants can protect bacterial cells during thedrying steps when water is removed. Some sugars, such as sucrose andtrehalose, can act as both cryoprotectants and lyoprotectants. Use ofskim milk can also provide protective effects. Other examples ofexcipients include glucose, maltose, lactose, mannitol, glycine,glycerol, sodium chloride, yeast extract, dextran, dextrose,polydextrose, monosodium glutamate, maltodextrin, antioxidants (e.g.,ascorbic acid), saccharides, disaccharides, sugars, and others. In oneexample, excipients used in a formulation include various combinationsof sucrose, trehalose, monosodium glutamate (MSG), recombinant humanserum albumin (rHSA), and amino acid mix. In a specific example, theexcipients comprise, consist essentially of, or consist of sucrose, suchas about 5% w/v (weight per volume) sucrose or about 2.5% w/v sucrose.For example, the formulation buffer can comprise about 1% to about 5%w/v sucrose, about 2% to about 3% w/v sucrose, or about 2.5% w/vsucrose.

Optionally, the excipients do not include one or more or all oftrehalose, MSG, rHSA, amino acid mix, skim milk, glucose, maltose,lactose, mannitol, glycine, glycerol, sodium chloride, yeast extract,dextran, dextrose, polydextrose, monosodium glutamate, maltodextrin,ascorbic acid, saccharides other than sucrose, disaccharides other thansucrose, sugars other than sucrose, or antioxidants. Optionally, theexcipients do not include one or more or all of trehalose, MSG, andrHSA.

The concentration of the bacteria or Listeria in the formulation can beany suitable concentration. For example, the concentration can bebetween about 1×10E9 and about 1×10E10 colony forming units (CFU) permilliliter.

C. Freezing Step

The first step in lyophilization is the freezing step. During thisstage, the formulation is cooled. This can be accomplished, for example,in a shelf freeze dryer by reducing the temperature of the lyophilizershelves (i.e., reducing the shelf temperature). During freezing, icecrystals are formed that can damage bacteria. The growth of the icecrystals is dependent on the freezing rate and temperature. In someembodiments, a higher freezing rate is utilized. A higher freezing ratercan lead to the formation of smaller ice crystals thus reducing cellulardamage as compared to a slower freezing rate. The formation of icecrystals can be detrimental to bacteria. As water crystallizes, thesolutes in the remaining unfrozen fraction concentrate, which can leadto chemical and osmotic damage. Although freezing bacteria at lowertemperatures corresponds to higher freezing rates and will result insmaller ice crystals, which should limit the cellular damage, a higherfreezing rate does not always corresponds with the best viabilityresults. Optimal freezing conditions can vary depending on protectantsused in the formulation and the strain of bacteria.

The holding temperature (e.g., the shelf temperature) for the freezingstep can be reached by reducing the temperature (e.g., the shelftemperature) at a rate of, for example, about 0.2° C. to about 2.0° C.per minute. Alternatively, the holding temperature (e.g., the shelftemperature) for the freezing step can be reached by reducing thetemperature (e.g., the shelf temperature) at a rate of, for example,about 0.2° C. to about 1.8° C. per minute, about 0.4° C. to about 1.6°C. per minute, about 0.6° C. to about 1.4° C. per minute, about 0.8° C.to about 1.2° C. per minute, or about 0.9° C. to about 1.1° C. perminute. For example, the temperature can be reduced to the freezingtemperature at a rate of about 0.2° C., about 0.3° C., about 0.4° C.,about 0.5° C., about 0.6° C., about 0.7° C., about 0.8° C., about 0.9°C., about 1.0° C., about 1.1° C., about 1.2° C., about 1.3° C., about1.4° C., about 1.5° C., about 1.6° C., about 1.7° C., about 1.8° C.,about 1.9° C., or about 2.0° C. per minute. In a specific example, theholding temperature for the freezing step is reached by decreasing thetemperature to the holding temperature at a rate of about 1° C. perminute.

The freezing step can be for any suitable time for freezing the bacteriaor Listeria strain. Likewise, the temperature can be held at thefreezing temperature for any suitable time for freezing the bacteria orListeria strain. For example, the freezing step can be, or thetemperature can be held at the freezing temperature, for about 2 toabout 6, about 2.5 to about 6, about 1 to about 6, about 1 to about 5,about 1 to about 4, about 1 to about 3, about 1 to about 2, about 1.5 toabout 2.5, about 1.5 to about 5.5, about 2 to about 5, about 2.5 toabout 4.5, about 3 to about 4, about 1, about 1.5, about 2, about 2.5,about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, or about 6hours. In a specific example, the freezing step can be or thetemperature can be held at the freezing temperature for 3.5 hours. Inanother specific example, freezing step can be or the temperature can beheld at the freezing temperature for 2 hours. In another specificexample, freezing step can be or the temperature can be held at thefreezing temperature for 1.5 hours. In another specific example, thetime of the entire freezing step (e.g., ramping the temperature to thefreezing temperature and then holding at the freezing temperature) isabout 3.5-4.5 hours or about 3.5 hours.

The freezing temperature (i.e., the holding temperature) can be anytemperature suitable for freezing the bacteria or Listeria strain. Insome embodiments, the freezing temperature (e.g., the shelf temperature)is such that the temperature of the formulation is below the glasstransition temperature of the solution, which in the case of sucroseformulations can be, for example, about −32° C. Temperatures above thistemperature may not truly freeze the solution, which may then undergocollapse during lyophilization, potentially leading to loss ofviability. For example, the temperature (e.g., the shelf temperature)can be between about −49° C. to about −25° C., about −47° C. to about−40° C., about −45° C. to about −35° C., about −10° C. to about −80° C.,about −15° C. to about −75° C., about −20° C. to about −70° C., about−25° C. to about −65° C., about −30° C. to about −60° C., about −35° C.to about −55° C., about −40° C. to about −50° C., about −41° C. to about−49° C., about −42° C. to about −48° C., about −43° C. to about −47° C.,or about −44° C. to about −46° C. In a specific example, the freezingtemperature can be about −45° C. In another example, the temperature canbe between about −49° C. to about −32° C., about −47° C. to about −40°C., about −45° C. to about −35° C., about −32° C. to about −80° C.,about −32° C. to about −75° C., about −32° C. to about −70° C., about−32° C. to about −65° C., about −32° C. to about −60° C., −49° C. toabout −33° C., about −33° C. to about −80° C., about −33° C. to about−75° C., about −33° C. to about −70° C., about −33° C. to about −65° C.,about −33° C. to about −60° C., about −35° C. to about −55° C., about−40° C. to about −50° C., about −41° C. to about −49° C., about −42° C.to about −48° C., about −43° C. to about −47° C., or about −44° C. toabout −46° C. In one example, the freezing temperature can be about −39°C. In another example, the freezing temperature can be about −45° C. Ina specific example, the holding temperature in the freezing step isbetween about −40° C. and about −50° C. (e.g., about −45° C.), thefreezing step comprises decreasing the temperature to the holdingtemperature at a rate of about 1° C. per minute, and the cooling in thefreezing step is from about 2 hours to about 4 hours (e.g., the freezingstep comprises holding the composition at the holding temperature forabout 2 hours).

D. Primary Drying Step

The second step in lyophilization is the primary drying step. In theprimary drying step, exposing the composition comprising the bacteria orListeria strain produced by the freezing step to a vacuum at anincreased temperature. In this step, the frozen water is removed bysublimation under vacuum.

The temperature for the primary drying step can be reached by increasingthe temperature (e.g., the shelf temperature) at a rate of, for example,about 0.2° C. to about 2.0° C. per minute. Alternatively, the holdingtemperature (e.g., the shelf temperature) for the primary drying stepcan be reached by increasing the temperature (e.g., the shelftemperature) at a rate of, for example, about 0.2° C. to about 1.8° C.per minute, about 0.4° C. to about 1.6° C. per minute, about 0.6° C. toabout 1.4° C. per minute, about 0.8° C. to about 1.2° C. per minute, orabout 0.9° C. to about 1.1° C. per minute. For example, the temperaturecan be increased to the primary drying temperature at a rate of about0.2° C., about 0.3° C., about 0.4° C., about 0.5° C., about 0.6° C.,about 0.7° C., about 0.8° C., about 0.9° C., about 1.0° C., about 1.1°C., about 1.2° C., about 1.3° C., about 1.4° C., about 1.5° C., about1.6° C., about 1.7° C., about 1.8° C., about 1.9° C., or about 2.0° C.per minute. In a specific example, the holding temperature for theprimary drying step is reached by increasing the temperature to theholding temperature at a rate of about 1° C. per minute.

The primary drying step can be for any suitable time. Likewise, theholding temperature (e.g., the shelf temperature) can be held at theprimary drying temperature for any suitable time. The temperature shouldbe held at the primary drying temperature until primary drying iscompleted. This time can vary depending on the lyophilizer, the vialsize, the fill volume, the number of vials, the pressure, and othervariables. The end of primary drying can be determined, for example,when the product temperature rises to a value at or above the shelftemperature. It can also be determined, for example, by a pressure risetest in which the freeze drying chamber is isolated from the vacuum pumpto determine how much the pressure rises due to continued watersublimation. For example, the primary drying step can be or thetemperature can be held at the primary drying temperature for about 10to about 29, about 29 to about 42, about 36, about 10 to about 80, about10 to about 70, about 10 to about 60, about 10 to about 50, about 10 toabout 40, about 10 to about 30, or about 20 to about 30 hours. In aspecific example, primary drying step can be or the temperature can beheld at the primary drying temperature for about 25 to about 35, about26 to about 34, about 27 to about 33, about 28 to about 32, about 29 toabout 31, or about 30 hours. In another specific example, primary dryingstep can be or the temperature can be held at the primary dryingtemperature for about 20 to about 30, about 21 to about 30, about 22 toabout 30, about 23 to about 29, about 24 to about 28, about 25 to about27, or about 26 hours.

The primary drying step can be or the temperature can be held at theprimary drying holding temperature for a time period defined as about 8to about 20, about 9 to about 19, about 10 to about 18, about 11 toabout 17, about 12 to about 16, about 13 to about 15, or about 14 hoursafter a probe in the lyophilizer (e.g., a probe for cold spots in thelyophilizer, such as in the center of the lyophilizer) has crossed theprimary drying holding temperature or the T_(s) set point (e.g., ofabout −18° C.). Alternatively, the drying step can be or the temperaturecan be held at the primary drying holding temperature for a time perioddefined as about 8 to about 20, about 9 to about 19, about 10 to about18, about 11 to about 17, about 12 to about 16, about 13 to about 15, orabout 14 hours after the composition being lyophilized (e.g., thesamples of the compositions in the cold spots of the lyophilizer, or allsamples of the compositions in the lyophilizer) has reached the primarydrying holding temperature or the T_(s) set point (e.g., about −18° C.).In a specific example, the primary drying step can be or the temperaturecan be held at the primary drying holding temperature for a time perioddefined as about 14 hours after a probe in the lyophilizer (e.g., aprobe for cold spots in the lyophilizer, such as in the center of thelyophilizer) has crossed the primary drying holding temperature or theT_(s) set point (e.g., about −18° C.) or after the composition beinglyophilized (e.g., the samples of the compositions in the cold spots ofthe lyophilizer, or all samples of the compositions in the lyophilizer)has reached the primary drying holding temperature or the T_(s) setpoint (e.g., about −18° C.), which can be, for example, about 30 hours.

The end of the primary drying step can be about 8 to about 20, about 9to about 19, about 10 to about 18, about 11 to about 17, about 12 toabout 16, about 13 to about 15, or about 14 hours after a probe in thelyophilizer (e.g., a probe for cold spots in the lyophilizer, such as inthe center of the lyophilizer) has crossed the primary drying holdingtemperature or the T_(s) set point (e.g., about −18° C.). Alternatively,the end of the primary drying step can be about 8 to about 20, about 9to about 19, about 10 to about 18, about 11 to about 17, about 12 toabout 16, about 13 to about 15, or about 14 hours after the compositionbeing lyophilized (e.g., the samples of the compositions in the coldspots of the lyophilizer, or all samples of the compositions in thelyophilizer) has reached the primary drying holding temperature or theT_(s) set point (e.g., of about −18° C.). In a specific example, the endof the primary drying step can be about 14 hours after a probe in thelyophilizer (e.g., a probe for cold spots in the lyophilizer, such as inthe center of the lyophilizer) has crossed the primary drying holdingtemperature or the T_(s) set point (e.g., about −18° C.) or after thecomposition being lyophilized (e.g., the samples of the compositions inthe cold spots of the lyophilizer, or all samples of the compositions inthe lyophilizer) has reached the primary drying holding temperature orthe T_(s) set point (e.g., of about −18° C.), which can be, for example,about 30 hours.

The primary drying temperature (e.g., the shelf temperature or theholding temperature) can be any temperature suitable for drying thebacteria or Listeria strain. For example, the holding temperature can bebetween about 0° C. to about −30° C., 0° C. to about −19° C., about −5°C. to about −30° C., about −10° C. to about −25° C., about −15° C. toabout −20° C., about −17° C. to about −19° C., about −12° C. to about−30° C., about −12° C. to about −24° C., about −12° C. to about −22° C.,about −14° C. to about −22° C., about −15° C. to about −21° C., about−16° C. to about −20° C., about −17° C. to about −19° C., about −18° C.to about −22° C., about −30°, about −29°, about −28°, about −27°, about−26°, about −25°, about −24°, about −23°, about −22°, about −21°, about−20°, about −19°, about −18°, about −17°, about −16°, about −15°, about−14°, about −13°, about −12°, about −11°, about −10°, about −9°, about−8°, about −7°, about −6°, about −5°, about −4°, about −3°, about −2°,about −1°, or about 0°. For example, the temperature can be no more thanabout −30°, about −29°, about −28°, about −27°, about −26°, about −25°,about −24°, about −23°, about −22°, about −21°, about −20°, about −19°,about −18°, about −17°, about −16°, about −15°, about −14°, about −13°,about −12°, about −11°, or about −10° C. In a specific example, theprimary drying temperature can be about −25° C. to about −35° C., about−26° C. to about −37° C., about −27° C. to about −33° C., about −28° C.to about −32° C., about −29° C. to about −31° C., or about −30° C. In aspecific example, the primary drying temperature can be about −17° C. toabout −27° C., about −18° C. to about −26° C., about −19° C. to about−25° C., about −20° C. to about −24° C., about −21° C. to about −23° C.,or about −22° C. In another specific example, the primary dryingtemperature can be about −7° C. to about −17° C., about −8° C. to about−16° C., about −9° C. to about −15° C., about −10° C. to about −14° C.,about −11° C. to about −13° C., or about −12° C. In another specificexample, the primary drying temperature can be about −13° C. to about−23° C., about −14° C. to about −22° C., about −15° C. to about −21° C.,about −16° C. to about −20° C., about −17° C. to about −19° C., or about−18° C. In a specific example, the holding temperature in the primarydrying step is between about −17° C. to about −19° C., or about −18° C.

The pressure (vacuum conditions) can be any suitable pressure. In somecases, the pressure should be no more than 50% of vapor pressure of iceat the glass transition temperature of the formulation (e.g., about0.270 mbar). It should also not be too low. For example, the pressurecan be from about 0.140 to about 0.050, about 0.100 to about 0.060,about 0.100 to about 0.070, about 0.100 to about 0.080, about 0.099 toabout 0.081, about 0.098 to about 0.082, about 0.097 to about 0.083,about 0.096 to about 0.084, about 0.095 to about 0.085, about 0.094 toabout 0.086, about 0.093 to about 0.087, about 0.092 to about 0.088,about 0.091 to about 0.089, about 0.090 mbar, or about 0.120 mbar. In aspecific example, the pressure is about 0.090 mbar.

In a specific example, the holding temperature in the primary dryingstep is between about −17° C. and about −19° C. (e.g., about −18° C.),the primary drying step comprises increasing the temperature to theholding temperature at a rate of about 1° C. per minute, and the primarydrying step is from about 10 hours to about 40 hours (e.g., about 20 toabout 40 hours, or about 25-35 hours, such as about 30 hours or about 32hours).

E. Secondary Drying Step

The third step in lyophilization is the secondary drying step. In thesecondary drying step, exposing the composition comprising the bacteriaor Listeria strain produced by the primary drying step to a vacuum at anincreased temperature. In this step, the unfrozen water is removed bydesorption.

The temperature for the secondary drying step can be reached byincreasing the temperature (e.g., the shelf temperature) at a rate of,for example, about 0.2° C. to about 2.0° C. per minute. Alternatively,the holding temperature (e.g., the shelf temperature) for the secondarydrying step can be reached by increasing the temperature (e.g., theshelf temperature) at a rate of, for example, about 0.2° C. to about1.8° C. per minute, about 0.2° C. to about 1.6° C. per minute, about0.2° C. to about 1.4° C. per minute, about 0.2° C. to about 1.2° C. perminute, about 0.2° C. to about 1.0° C. per minute, about 0.2° C. toabout 0.8° C. per minute, about 0.2° C. to about 0.6° C. per minute,about 0.2° C. to about 0.4° C. per minute. For example, the temperaturecan be increased to the secondary drying temperature at a rate of about0.2° C., about 0.3° C., about 0.4° C., about 0.5° C., about 0.6° C.,about 0.7° C., about 0.8° C., about 0.9° C., about 1.0° C., about 1.1°C., about 1.2° C., about 1.3° C., about 1.4° C., about 1.5° C., about1.6° C., about 1.7° C., about 1.8° C., about 1.9° C., or about 2.0° C.per minute. In a specific example, the holding temperature for thesecondary drying step is reached by increasing the temperature to theholding temperature at a rate of about 0.2° C. per minute.

The secondary drying step can be for any suitable time. Likewise, thetemperature (e.g., the shelf temperature or the holding temperature) canbe held at the secondary drying temperature for any suitable time. Forexample, the temperature can be held at the secondary drying temperaturefor any suitable time to achieve the desired residual moisture levels inthe lyophilized product. For example, the secondary drying step can beor the temperature can be held at the secondary drying temperature forabout 5 to about 40, about 10 to about 30, about 15 to about 25, about 2to about 25, about 2 to about 20, about 2 to about 10, about 2 to about4, about 1 to about 25, about 1 to about 20, about 1 to about 10, about1 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about6, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1to about 2, about 1.5 to about 2.5, about 2.5 to about 3.5, about 15,about 14, about 13, about 12, about 11, about 10, about 9, about 8,about 7, about 6, about 5, about 4, about 3, about 2, or about 1 hours.In a specific example, secondary drying step can be or the secondarydrying hold time is no more than 10 hours. In another specific example,the secondary drying hold time is no more than 6 hours. In anotherspecific example, the temperature can be held at the secondary dryingtemperature for about 3 hours. In another specific example, thetemperature can be held at the secondary drying temperature for about 2hours. In one example, the secondary drying step is for about 1 hour toabout 10 hours. In another example, the secondary drying step comprisesholding the composition at the holding temperature for about 2 hours toabout 6 hours, for about 5 hours to about 6 hours, or for about 5 hoursor about 6 hours.

The secondary drying temperature (e.g., the shelf temperature or theholding temperature) can be any temperature suitable for drying thebacteria or Listeria strain to achieve the desired residual moisturelevels in the lyophilized product. For example, the temperature can bebetween about 5° C. to about 40° C., about 5° C. to about 30° C., about10° C. to about 30° C., about 20° C. to about 30° C., or about 15° C. toabout 25° C. In a specific example, the secondary drying temperature canbe about 25° C. In another specific example, the secondary dryingtemperature can be about 20° C. In another specific example, thesecondary drying temperature is no more than about 20° C. In anotherexample, the temperature can be between about 5° C. to about 20° C.,about 9° C. to about 15° C., about 10° C. to about 15° C., about 11° C.to about 14° C., about 11° C. to about 13° C., about 5° C., about 6° C.,about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about12° C., about 13° C., about 14° C., about 15° C., about 16° C., about17° C., about 18° C., about 19° C., or about 20° C. In a specificexample, the secondary drying temperature can be about 12° C.Alternatively, the holding temperature can be between about −10° C. andabout 30° C., between about −10° C. and about 25° C., between about −10°C. and about 20° C., between about −10° C. and about 10° C., betweenabout −5° C. and about 30° C., between about −5° C. and about 25° C.,between about −5° C. and about 20° C., between about −5° C. and about15° C., between about −5° C. and about 10° C., between about −5° C. andabout 5° C., between about −4° C. and about 4° C., between about −3° C.and about 3° C., between about −2° C. and about 2° C., between about −1°C. and about 1° C., or about 0° C. In a specific example, the holdingtemperature can be between about −5° C. and about 5° C. or about 0° C.

The pressure (vacuum conditions) can be any suitable pressure. In somecases, the pressure is in the same range as for the primary drying step.However, some cycles during secondary drying may have full vacuum. Forexample, the pressure can be from about 0.140 to about 0.020, 0.140 toabout 0.030, 0.140 to about 0.040, 0.140 to about 0.050, about 0.100 toabout 0.060, about 0.100 to about 0.070, about 0.100 to about 0.080,about 0.099 to about 0.081, about 0.098 to about 0.082, about 0.097 toabout 0.083, about 0.096 to about 0.084, about 0.095 to about 0.085,about 0.094 to about 0.086, about 0.093 to about 0.087, about 0.092 toabout 0.088, about 0.091 to about 0.089, about 0.090 mbar, or about0.120 mbar. In a specific example, the pressure is about 0.090 mbar.

In a specific example, the holding temperature in the secondary dryingstep is between about −5° C. and about 5° C. (e.g., about 0° C.), thesecondary drying step comprises increasing the temperature to theholding temperature at a rate of about 0.2° C. per minute, and thesecondary drying step secondary drying step comprises holding thecomposition at the holding temperature for about 5 hours to about 6hours.

The secondary drying step can result in a lyophilized product having anydesired residual moisture. For example, the residual moisture can be nomore than about 7.0%, about 6.9%, about 6.8%, about 6.7%, about 6.6%,about 6.5%, about 6.4%, about 6.3%, about 6.2%, about 6.1%, about 6.0%,about 5.9%, about 5.8%, about 5.7%, about 5.6%, about 5.5%, about 5.4%,about 5.3%, about 5.2%, about 5.1%, about 5.0%, about 4.9%, about 4.8%,about 4.7%, about 4.6%, about 4.5%, about 4.4%, about 4.3%, about 4.2%,about 4.1%, about 4.0%, about 3.9%, about 3.8%, about 3.7%, about 3.6%,about 3.5%, about 3.4%, about 3.3%, about 3.2%, about 3.1%, about 3.0%,about 2.9%, about 2.8%, about 2.7%, about 2.6%, about 2.5%, about 2.4%,about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%,about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%,about 1.1%, or about 1.0%. Alternatively, the residual moisture can beat least about 7.0%, about 6.9%, about 6.8%, about 6.7%, about 6.6%,about 6.5%, about 6.4%, about 6.3%, about 6.2%, about 6.1%, about 6.0%,about 5.9%, about 5.8%, about 5.7%, about 5.6%, about 5.5%, about 5.4%,about 5.3%, about 5.2%, about 5.1%, about 5.0%, about 4.9%, about 4.8%,about 4.7%, about 4.6%, about 4.5%, about 4.4%, about 4.3%, about 4.2%,about 4.1%, about 4.0%, about 3.9%, about 3.8%, about 3.7%, about 3.6%,about 3.5%, about 3.4%, about 3.3%, about 3.2%, about 3.1%, about 3.0%,about 2.9%, about 2.8%, about 2.7%, about 2.6%, about 2.5%, about 2.4%,about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%,about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%,about 1.1%, or about 1.0%. In a specific example, the residual moisturecan be at least about 1%, at least about 1.5%, or at about least 2% andno more than about 7%. Alternatively, the residual moisture can bebetween about 1% to about 7%, about 1% to about 6.5%, about 1% to about6%, about 1% to about 5.5%, about 1% to about 5%, about 1.5% to about7%, about 1.5% to about 6.5%, about 1.5% to about 6%, about 1.5% toabout 5.5%, about 1.5% to about 5%, about 1.5% to about 4.5%, about 2%to about 7%, about 2% to about 6.5%, about 2% to about 6%, about 2% toabout 5.5%, about 2% to about 5%, about 2% to about 4.5%, about 2% toabout 4%, about 2% to about 3%, or about 3% to about 4%. In a specificexample, the residual moisture can be about 3% to about 4%, about 3.1%to about 3.9%, about 3.2% to about 3.8%, about 3.3% to about 3.7%, about3.4% to about 3.6%, or about 3.5%. In a specific example, the residualmoisture is at least about 2%, at least about 2.5%, or at least about3%. In another specific example, the residual moisture is between about1% and about 5%, between about 2% and about 4%, between about 2.5% andabout 3.5%, between about 2.5% and about 4%, between about 3% and about4%, or between about 3% and about 3.5%.

F. Storage and Reconstitution of Lyophilized Bacteria or Listeria

The resulting lyophilized bacteria or Listeria can be a lyophilizedcomposition comprising any combination of the components listed in theformulation section. In one example, the lyophilized compositioncomprises a Listeria strain, a buffer (e.g., a phosphate), and anexcipient (e.g., sucrose). Optionally, the lyophilized composition doesnot comprise one or more or all of trehalose, monosodium glutamate(MSG), and recombinant human serum albumin (rHSA). Optionally, thelyophilized composition does not comprise one or more or all of theoptional components listed in the formulation section.

The resulting lyophilized bacteria or Listeria can be a lyophilizedcomposition with any of the residual moisture levels listed elsewhereherein. As one example, the residual moisture level can be between about1% and about 5%, between about 2% and about 4%, or between about 3% andabout 4%.

The lyophilized bacteria can be stored under any suitable conditions,including any suitable temperature, relative humidity, and atmosphericoxygen level, which are well-known. The lyophilized bacteria or Listeriacan exhibit viability upon reconstitution of at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, or about 95% after storage for a defined amount of time. Thereconstitution can follow storage of the lyophilized bacteria orListeria for example, for 2 days, 3 days, 4 days, 1 week, 2 weeks, 3weeks, 1 month, 2 months, 3 months, 5 months, 6 months, 9 months, 12months (1 year), 15 months, 18 months, 21 months, or 24 months (2years).

The storage temperature of the lyophilized bacteria or Listeria can befor example, between about 0° C. and about 10° C., about 1° C. and about9° C., about 2° C. and about 8° C., about 2° C. and about 6° C., orabout 3° C. and about 5° C. In a specific example, the storagetemperature of can be between about 2° C. and about 8° C., or thestorage temperature can be about 4° C. In another example, the storagetemperature can be between about −15° C. and about −25° C., about −16°C. and about −24° C., about −17° C. and about −23° C., about −18° C. andabout −22° C., or about −19° C. and about −21° C. In a specific example,the storage temperature of can be about −20° C.

For example, the lyophilized bacteria or Listeria can show at leastabout 60%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or about100% viability after storage at about 2-8° C. (e.g., 4° C.) or about−20° C. for about 6 months, about 9 months, about 12 months, about 18months, or about 24 months. The lyophilized bacteria or Listeria canshow at least about 60%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or about 100% viability after storage at about 30° C., at aboutroom temperature (i.e., about 20-25° C. (e.g., 20° C., 21° C., 22° C.,23° C., 24° C., or 25° C.)), at about 2-8° C. (e.g., 4° C.) or about−20° C. for about 1 month, about 2 months, about 3 months, about 4months, about 5 months, about 6 months, about 9 months, about 12 months,about 18 months, or about 24 months. As one example, the lyophilizedbacteria or Listeria can show at least about 75% to about 80% viabilityat 2-8° C. after 6 months. As another example, the lyophilized bacteriaor Listeria can show at least about 95% to about 100% viability at −20°C. after 9 months. As another example, the lyophilized bacteria orListeria can show at least about 80% to about 90% viability at roomtemperature or at 30° C. after 2 months. As another example, thelyophilized bacteria or Listeria can show at least about 60%, 65%, 70%,75%, 80%, 85%, or 90% viability at about −20° C. after about 12 months,18 months, or 24 months. As another example, the lyophilized bacteria orListeria can show at least about 60%, 65%, 70%, 75%, 80% viability atabout 2-8° C. after about 12 months, 18 months, or 24 months. As anotherexample, the lyophilized bacteria or Listeria can show at least about60%, 65%, 70%, 75%, or 80% viability at about 2-8° C. after about 12months, 18 months, or 24 months.

After storage, the lyophilized bacteria or Listeria strain canoptionally be reconstituted with a solvent or diluent (e.g., water). Asone example, the solvent or diluent can be appropriate media forculturing the bacteria or Listeria strain. Methods for reconstitutionand rehydration of lyophilized bacteria or Listeria strains arewell-known. In one example, the volume of solvent used is the volume ofpre-lyophilization solution used to make the lyophilized bacteria orListeria strain. In another example, the volume of solvent used is morethan the volume of pre-lyophilization solution used to make thelyophilized bacteria or Listeria strain. In another example, the volumeof solvent used is less than the volume of pre-lyophilization solutionused to make the lyophilized bacteria or Listeria strain.

The reconstitution time can be any suitable reconstitution time. Forexample, the reconstitution time can be less than about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, or 30 minutes. In a specific example, thereconstitution time is less than about 2 minutes.

III. Recombinant Bacteria or Listeria Strains

The lyophilized compositions disclosed herein and the compositions thatundergo the lyophilization methods disclosed herein comprise bacteriastrains, such as a Listeria strain. Such bacteria strains can berecombinant bacteria strains. Such recombinant bacteria strains cancomprise a recombinant fusion polypeptide disclosed herein or a nucleicacid encoding the recombinant fusion polypeptide as disclosed elsewhereherein. In some embodiments, the bacteria strain is a Listeria strain,such as a Listeria monocytogenes (Lm) strain. Lm has a number ofinherent advantages as a vaccine vector. The bacterium grows veryefficiently in vitro without special requirements, and it lacks LPS,which is a major toxicity factor in gram-negative bacteria, such asSalmonella. Genetically attenuated Lm vectors also offer additionalsafety as they can be readily eliminated with antibiotics, in case ofserious adverse effects, and unlike some viral vectors, no integrationof genetic material into the host genome occurs.

The recombinant Listeria strain can be any Listeria strain. Examples ofsuitable Listeria strains include Listeria seeligeri, Listeria grayi,Listeria ivanovii, Listeria murrayi, Listeria welshimeri, Listeriamonocytogenes (Lm), or any other known Listeria species. In someembodiments, the recombinant listeria strain is a strain of the speciesListeria monocytogenes. Examples of Listeria monocytogenes strainsinclude the following: L. monocytogenes 10403S wild type (see, e.g.,Bishop and Hinrichs (1987) J Immunol 139:2005-2009; Lauer et al. (2002)J Bact 184:4177-4186); L. monocytogenes DP-L4056, which is phage cured(see, e.g., Lauer et al. (2002) J Bact 184:4177-4186); L. monocytogenesDP-L4027, which is phage cured and has an hly gene deletion (see, e.g.,Lauer et al. (2002) J Bact 184:4177-4186; Jones and Portnoy (1994)Infect Immunity 65:5608-5613); L. monocytogenes DP-L4029, which is phagecured and has an actA gene deletion (see, e.g., Lauer et al. (2002) JBact 184:4177-4186; Skoble et al. (2000) J Cell Biol 150:527-538); L.monocytogenes DP-L4042 (delta PEST) (see, e.g., Brockstedt et al. (2004)Proc Natl Acad Sci. USA 101:13832-13837 and supporting information); L.monocytogenes DP-L4097 (LLO-S44A) (see, e.g., Brockstedt et al. (2004)Proc Natl Acad Sci USA 101:13832-13837 and supporting information); L.monocytogenes DP-L4364 (delta lplA; lipoate protein ligase) (see, e.g.,Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837 andsupporting information); L. monocytogenes DP-L4405 (delta inlA) (see,e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837and supporting information); L. monocytogenes DP-L4406 (delta inlB)(see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA101:13832-13837 and supporting information); L. monocytogenes CS-L0001(delta actA; delta inlB) (see, e.g., Brockstedt et al. (2004) Proc NatlAcad Sci USA 101:13832-13837 and supporting information); L.monocytogenes CS-L0002 (delta actA; delta lplA) (see, e.g., Brockstedtet al. (2004) Proc Natl Acad Sci USA 101:13832-13837 and supportinginformation); L. monocytogenes CS-L0003 (LLO L461T; delta lplA) (see,e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA 101:13832-13837and supporting information); L. monocytogenes DP-L4038 (delta actA; LLOL461T) (see, e.g., Brockstedt et al. (2004) Proc Natl Acad Sci USA101:13832-13837 and supporting information); L. monocytogenes DP-L4384(LLO S44A; LLO L461T) (see, e.g., Brockstedt et al. (2004) Proc NatlAcad Sci USA 101:13832-13837 and supporting information); a L.monocytogenes strain with an lplA1 deletion (encoding lipoate proteinligase LplA1) (see, e.g., O'Riordan et al. (2003) Science 302:462-464);L. monocytogenes DP-L4017 (10403S with LLO L461T) (see, e.g., U.S. Pat.No. 7,691,393); L. monocytogenes EGD (see, e.g., GenBank Accession No.AL591824). In another embodiment, the Listeria strain is L.monocytogenes EGD-e (see GenBank Accession No. NC_003210; ATCC AccessionNo. BAA-679); L. monocytogenes DP-L4029 (actA deletion, optionally incombination with uvrAB deletion (DP-L4029uvrAB) (see, e.g., U.S. Pat.No. 7,691,393); L. monocytogenes actA-linlB—double mutant (see, e.g.,ATCC Accession No. PTA-5562); L. monocytogenes lplA mutant or hly mutant(see, e.g., US 2004/0013690); L. monocytogenes dal/dat double mutant(see, e.g., US 2005/0048081). Other L. monocytogenes strains includesthose that are modified (e.g., by a plasmid and/or by genomicintegration) to contain a nucleic acid encoding one of, or anycombination of, the following genes: hly (LLO; listeriolysin); iap(p60); inlA; inlB; inlC; dal (alanine racemase); dat (D-amino acidaminotransferase); plcA; plcB; actA; or any nucleic acid that mediatesgrowth, spread, breakdown of a single walled vesicle, breakdown of adouble walled vesicle, binding to a host cell, or uptake by a host cell.Each of the above references is herein incorporated by reference in itsentirety for all purposes.

The recombinant bacteria or Listeria can have wild-type virulence, canhave attenuated virulence, or can be avirulent. For example, arecombinant Listeria of can be sufficiently virulent to escape thephagosome or phagolysosome and enter the cytosol. Such Listeria strainscan also be live-attenuated Listeria strains, which comprise at leastone attenuating mutation, deletion, or inactivation as disclosedelsewhere herein. In some embodiments, the recombinant Listeria is anattenuated auxotrophic strain. An auxotrophic strain is one that isunable to synthesize a particular organic compound required for itsgrowth. Examples of such strains are described in U.S. Pat. No.8,114,414, herein incorporated by reference in its entirety for allpurposes.

In some embodiments, the recombinant Listeria strain lacks antibioticresistance genes. For example, such recombinant Listeria strains cancomprise a plasmid that does not encode an antibiotic resistance gene.However, some recombinant Listeria strains provided herein comprise aplasmid comprising a nucleic acid encoding an antibiotic resistancegene. Antibiotic resistance genes may be used in the conventionalselection and cloning processes commonly employed in molecular biologyand vaccine preparation. Exemplary antibiotic resistance genes includegene products that confer resistance to ampicillin, penicillin,methicillin, streptomycin, erythromycin, kanamycin, tetracycline,chloramphenicol (CAT), neomycin, hygromycin, and gentamicin.

A. Bacteria or Listeria Strains Comprising Recombinant FusionPolypeptides or Nucleic Acids Encoding Recombinant Fusion Polypeptides

The recombinant bacteria strains (e.g., Listeria strains) disclosedherein comprise a recombinant fusion polypeptide disclosed herein or anucleic acid encoding the recombinant fusion polypeptide as disclosedelsewhere herein.

In bacteria or Listeria strains comprising a nucleic acid encoding arecombinant fusion protein, the nucleic acid can be codon optimized.Examples of optimal codons utilized by L. monocytogenes for each aminoacid are shown US 2007/0207170, herein incorporated by reference in itsentirety for all purposes. A nucleic acid is codon-optimized if at leastone codon in the nucleic acid is replaced with a codon that is morefrequently used by L. monocytogenes for that amino acid than the codonin the original sequence.

The nucleic acid can be present in an episomal plasmid within thebacteria or Listeria strain and/or the nucleic acid can be genomicallyintegrated in the bacteria or Listeria strain. Some recombinant bacteriaor Listeria strains comprise two separate nucleic acids encoding tworecombinant fusion polypeptides as disclosed herein: one nucleic acid inan episomal plasmid, and one genomically integrated in the bacteria orListeria strain.

The episomal plasmid can be one that is stably maintained in vitro (incell culture), in vivo (in a host), or both in vitro and in vivo. If inan episomal plasmid, the open reading frame encoding the recombinantfusion polypeptide can be operably linked to a promoter/regulatorysequence in the plasmid. If genomically integrated in the bacteria orListeria strain, the open reading frame encoding the recombinant fusionpolypeptide can be operably linked to an exogenous promoter/regulatorysequence or to an endogenous promoter/regulatory sequence. Examples ofpromoters/regulatory sequences useful for driving constitutiveexpression of a gene are well-known and include, for example, an hly,hlyA, actA, prfA, and p60 promoters of Listeria, the Streptococcus bacpromoter, the Streptomyces griseus sgiA promoter, and the B.thuringiensis phaZ promoter. In some cases, an inserted gene of interestis not interrupted or subjected to regulatory constraints which oftenoccur from integration into genomic DNA, and in some cases, the presenceof the inserted heterologous gene does not lead to rearrangement orinterruption of the cell's own important regions.

Such recombinant bacteria or Listeria strains can be made bytransforming a bacteria or Listeria strain or an attenuated bacteria orListeria strain described elsewhere herein with a plasmid or vectorcomprising a nucleic acid encoding the recombinant fusion polypeptide.The plasmid can be an episomal plasmid that does not integrate into ahost chromosome. Alternatively, the plasmid can be an integrativeplasmid that integrates into a chromosome of the bacteria or Listeriastrain. The plasmids used herein can also be multicopy plasmids. Methodsfor transforming bacteria are well-known, and include calcium-chloridecompetent cell-based methods, electroporation methods,bacteriophage-mediated transduction, chemical transformation techniques,and physical transformation techniques. See, e.g., de Boer et al. (1989)Cell 56:641-649; Miller et al. (1995) FASEB J. 9:190-199; Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York; Ausubel et al. (1997) Current Protocols inMolecular Biology, John Wiley & Sons, New York; Gerhardt et al., eds.,1994, Methods for General and Molecular Bacteriology, American Societyfor Microbiology, Washington, D.C.; and Miller, 1992, A Short Course inBacterial Genetics, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., each of which is herein incorporated by reference in itsentirety for all purposes.

Bacteria or Listeria strains with genomically integrated heterologousnucleic acids can be made, for example, by using a site-specificintegration vector, whereby the bacteria or Listeria comprising theintegrated gene is created using homologous recombination. Theintegration vector can be any site-specific integration vector that iscapable of infecting a bacteria or Listeria strain. Such an integrationvector can comprise, for example, a PSA attPP′ site, a gene encoding aPSA integrase, a U153 attPP′ site, a gene encoding a U153 integrase, anA118 attPP′ site, a gene encoding an A118 integrase, or any other knownattPP′ site or any other phage integrase.

Such bacteria or Listeria strains comprising an integrated gene can alsobe created using any other known method for integrating a heterologousnucleic acid into a bacteria or Listeria chromosome. Techniques forhomologous recombination are well-known, and are described, for example,in Baloglu et al. (2005) Vet Microbiol 109(1-2):11-17); Jiang et al.2005) Acta Biochim Biophys Sin (Shanghai) 37(1):19-24), and U.S. Pat.No. 6,855,320, each of which is herein incorporated by reference in itsentirety for all purposes.

Integration into a bacteria or Listerial chromosome can also be achievedusing transposon insertion. Techniques for transposon insertion arewell-known, and are described, for example, for the construction ofDP-L967 by Sun et al. (1990) Infection and Immunity 58: 3770-3778,herein incorporated by reference in its entirety for all purposes.Transposon mutagenesis can achieve stable genomic insertion, but theposition in the genome where the heterologous nucleic acids has beeninserted is unknown.

Integration into a bacterial or Listerial chromosome can also beachieved using phage integration sites (see, e.g., Lauer et al. (2002) JBacteriol 184(15):4177-4186, herein incorporated by reference in itsentirety for all purposes). For example, an integrase gene andattachment site of a bacteriophage (e.g., U153 or PSA listeriophage) canbe used to insert a heterologous gene into the corresponding attachmentsite, which may be any appropriate site in the genome (e.g. comK or the3′ end of the arg tRNA gene). Endogenous prophages can be cured from theutilized attachment site prior to integration of the heterologousnucleic acid. Such methods can result, for example, in single-copyintegrants. In order to avoid a “phage curing step,” a phage integrationsystem based on PSA phage can be used (see, e.g., Lauer et al. (2002) JBacteriol 184:4177-4186, herein incorporated by reference in itsentirety for all purposes). Maintaining the integrated gene can require,for example, continuous selection by antibiotics. Alternatively, aphage-based chromosomal integration system can be established that doesnot require selection with antibiotics. Instead, an auxotrophic hoststrain can be complemented. For example, a phage-based chromosomalintegration system for clinical applications can be used, where a hoststrain that is auxotrophic for essential enzymes, including, forexample, D-alanine racemase is used (e.g., Lm dal(−)dat(−)).

Conjugation can also be used to introduce genetic material and/orplasmids into bacteria. Methods for conjugation are well-known, and aredescribed, for example, in Nikodinovic et al. (2006) Plasmid56(3):223-227 and Auchtung et al. (2005) Proc Natl Acad Sci USA102(35):12554-12559, each of which is herein incorporated by referencein its entirety for all purposes.

In a specific example, a recombinant bacteria or Listeria strain cancomprise a nucleic acid encoding a recombinant fusion polypeptidegenomically integrated into the bacteria or Listeria genome as an openreading frame with an endogenous actA sequence (encoding an ActAprotein) or an endogenous hly sequence (encoding an LLO protein). Forexample, the expression and secretion of the fusion polypeptide can beunder the control of the endogenous actA promoter and ActA signalsequence or can be under the control of the endogenous hly promoter andLLO signal sequence. As another example, the nucleic acid encoding arecombinant fusion polypeptide can replace an actA sequence encoding anActA protein or an hly sequence encoding an LLO protein.

Selection of recombinant bacteria or Listeria strains can be achieved byany means. For example, antibiotic selection can be used. Antibioticresistance genes may be used in the conventional selection and cloningprocesses commonly employed in molecular biology and vaccinepreparation. Exemplary antibiotic resistance genes include gene productsthat confer resistance to ampicillin, penicillin, methicillin,streptomycin, erythromycin, kanamycin, tetracycline, chloramphenicol(CAT), neomycin, hygromycin, and gentamicin. Alternatively, auxotrophicstrains can be used, and an exogenous metabolic gene can be used forselection instead of or in addition to an antibiotic resistance gene. Asan example, in order to select for auxotrophic bacteria comprising aplasmid encoding a metabolic enzyme or a complementing gene providedherein, transformed auxotrophic bacteria can be grown in a medium thatwill select for expression of the gene encoding the metabolic enzyme(e.g., amino acid metabolism gene) or the complementing gene.Alternatively, a temperature-sensitive plasmid can be used to selectrecombinants or any other known means for selecting recombinants.

B. Attenuation of Bacteria or Listeria Strains

The recombinant bacteria strains (e.g., recombinant Listeria strains)disclosed herein can be attenuated. The term “attenuation” encompasses adiminution in the ability of the bacterium to cause disease in a hostanimal. For example, the pathogenic characteristics of an attenuatedListeria strain may be lessened compared with wild-type Listeria,although the attenuated Listeria is capable of growth and maintenance inculture. In some embodiments, using as an example the intravenousinoculation of BALB/c mice with an attenuated Listeria, the lethal doseat which 50% of inoculated animals survive (LD₅₀) is increased above theLD₅₀ of wild-type Listeria by at least about 10-fold, by at least about100-fold, by at least about 1,000 fold, by at least about 10,000 fold,or by at least about 100,000-fold. An attenuated strain of Listeria isthus one that does not kill an animal to which it is administered, or isone that kills the animal only when the number of bacteria administeredis vastly greater than the number of wild-type non-attenuated bacteriawhich would be required to kill the same animal. An attenuated bacteriumshould also be construed to mean one which is incapable of replicationin the general environment because the nutrient required for its growthis not present therein. Thus, the bacterium is limited to replication ina controlled environment wherein the required nutrient is provided.Attenuated strains are environmentally safe in that they are incapableof uncontrolled replication

(I) Methods of Attenuating Bacteria and Listeria Strains

Attenuation can be accomplished by any known means. For example, suchattenuated strains can be deficient in one or more endogenous virulencegenes or one or more endogenous metabolic genes. Examples of such genesare disclosed herein, and attenuation can be achieved by inactivation ofany one of or any combination of the genes disclosed herein.Inactivation can be achieved, for example, through deletion or throughmutation (e.g., an inactivating mutation). The term “mutation” includesany type of mutation or modification to the sequence (nucleic acid oramino acid sequence) and may encompass a deletion, a truncation, aninsertion, a substitution, a disruption, or a translocation. Forexample, a mutation can include a frameshift mutation, a mutation whichcauses premature termination of a protein, or a mutation of regulatorysequences which affect gene expression. Mutagenesis can be accomplishedusing recombinant DNA techniques or using traditional mutagenesistechnology using mutagenic chemicals or radiation and subsequentselection of mutants. In some embodiments, deletion mutants are usedbecause of the accompanying low probability of reversion. The term“metabolic gene” refers to a gene encoding an enzyme involved in orrequired for synthesis of a nutrient utilized or required by a hostbacteria. For example, the enzyme can be involved in or required for thesynthesis of a nutrient required for sustained growth of the hostbacteria. The term “virulence” gene includes a gene whose presence oractivity in an organism's genome that contributes to the pathogenicityof the organism (e.g., enabling the organism to achieve colonization ofa niche in the host (including attachment to cells), immunoevasion(evasion of host's immune response), immunosuppression (inhibition ofhost's immune response), entry into and exit out of cells, or obtainingnutrition from the host).

A specific example of such an attenuated strain is Listeriamonocytogenes (Lm) dal(−)dat(−) (Lmdd). Another example of such anattenuated strain is Lm dal(−)dat(−)ΔactA (LmddA). See, e.g., US2011/0142791, herein incorporated by references in its entirety for allpurposes. LmddA is based on a Listeria strain which is attenuated due tothe deletion of the endogenous virulence gene actA. Such strains canretain a plasmid for antigen expression in vivo and in vitro bycomplementation of the dal gene. Alternatively, the LmddA can be adal/dat/actA Listeria having mutations in the endogenous dal, dat, andactA genes. Such mutations can be, for example, a deletion or otherinactivating mutation.

Another specific example of an attenuated strain is Lm prfA(−) or astrain having a partial deletion or inactivating mutation in the prfAgene. The PrfA protein controls the expression of a regulon comprisingessential virulence genes required by Lm to colonize its vertebratehosts; hence the prfA mutation strongly impairs PrfA ability to activateexpression of PrfA-dependent virulence genes.

Yet another specific example of an attenuated strain is LminlB(−)actA(−) in which two genes critical to the bacterium's naturalvirulence—internalin B and act A—are deleted.

Other examples of attenuated bacteria or Listeria strains includebacteria or Listeria strains deficient in one or more endogenousvirulence genes. Examples of such genes include actA, prfA, plcB, plcA,inlA, inlB, inlC, inlJ, and bsh in Listeria. Attenuated Listeria strainscan also be the double mutant or triple mutant of any of theabove-mentioned strains. Attenuated Listeria strains can comprise amutation or deletion of each one of the genes, or comprise a mutation ordeletion of, for example, up to ten of any of the genes provided herein(e.g., including the actA, prfA, and dal/dat genes). For example, anattenuated Listeria strain can comprise a mutation or deletion of anendogenous internalin C (inlC) gene and/or a mutation or deletion of anendogenous actA gene. Alternatively, an attenuated Listeria strain cancomprise a mutation or deletion of an endogenous internalin B (inlB)gene and/or a mutation or deletion of an endogenous actA gene.Alternatively, an attenuated Listeria strain can comprise a mutation ordeletion of endogenous inlB, inlC, and actA genes. Translocation ofListeria to adjacent cells is inhibited by the deletion of theendogenous actA gene and/or the endogenous inlC gene or endogenous inlBgene, which are involved in the process, thereby resulting in highlevels of attenuation with increased immunogenicity and utility as astrain backbone. An attenuated Listeria strain can also be a doublemutant comprising mutations or deletions of both plcA and plcB. In somecases, the strain can be constructed from the EGD Listeria backbone.

A bacteria or Listeria strain can also be an auxotrophic strain having amutation in a metabolic gene. As one example, the strain can bedeficient in one or more endogenous amino acid metabolism genes. Forexample, the generation of auxotrophic strains of Listeria deficient inD-alanine, for example, may be accomplished in a number of ways that arewell-known, including deletion mutations, insertion mutations,frameshift mutations, mutations which cause premature termination of aprotein, or mutation of regulatory sequences which affect geneexpression. In some embodiments, deletion mutants are used because ofthe accompanying low probability of reversion of the auxotrophicphenotype. As an example, mutants of D-alanine which are generatedaccording to the protocols presented herein may be tested for theability to grow in the absence of D-alanine in a simple laboratoryculture assay. Those mutants which are unable to grow in the absence ofthis compound can be selected.

Examples of endogenous amino acid metabolism genes include a vitaminsynthesis gene, a gene encoding pantothenic acid synthase, a D-glutamicacid synthase gene, a D-alanine amino transferase (dat) gene, aD-alanine racemase (dal) gene, dga, a gene involved in the synthesis ofdiaminopimelic acid (DAP), a gene involved in the synthesis of Cysteinesynthase A (cysK), a vitamin-B12 independent methionine synthase, trpA,trpB, trpE, asnB, gltD, gltB, leuA, argG, and thrC. The Listeria straincan be deficient in two or more such genes (e.g., dat and dal).D-glutamic acid synthesis is controlled in part by the dal gene, whichis involved in the conversion of D-glu+pyr to alpha-ketoglutarate+D-ala,and the reverse reaction.

As another example, an attenuated Listeria strain can be deficient in anendogenous synthase gene, such as an amino acid synthesis gene. Examplesof such genes include folP, a gene encoding a dihydrouridine synthasefamily protein, ispD, ispF, a gene encoding a phosphoenolpyruvatesynthase, hisF, hisH, fliI, a gene encoding a ribosomal large subunitpseudouridine synthase, ispD, a gene encoding a bifunctional GMPsynthase/glutamine amidotransferase protein, cobS, cobB, cbiD, a geneencoding a uroporphyrin-III C-methyltransferase/uroporphyrinogen-IIIsynthase, cobQ, uppS, truB, dxs, mvaS, dapA, ispG, folC, a gene encodinga citrate synthase, argJ, a gene encoding a 3-deoxy-7-phosphoheptulonatesynthase, a gene encoding an indole-3-glycerol-phosphate synthase, agene encoding an anthranilate synthase/glutamine amidotransferasecomponent, menB, a gene encoding a menaquinone-specific isochorismatesynthase, a gene encoding a phosphoribosylformylglycinamidine synthase Ior II, a gene encoding a phosphoribosylaminoimidazole-succinocarboxamidesynthase, carB, carA, thyA, mgsA, aroB, hepB, rluB, ilvB, ilvN, alsS,fabF, fabH, a gene encoding a pseudouridine synthase, pyrG, truA, pabB,and an ATP synthase gene (e.g., atpC, atpD-2, aptG, atpA-2, and soforth).

Attenuated Listeria strains can be deficient in endogenous phoP, aroA,aroC, aroD, or plcB. As yet another example, an attenuated Listeriastrain can be deficient in an endogenous peptide transporter. Examplesinclude genes encoding an ABC transporter/ATP-binding/permease protein,an oligopeptide ABC transporter/oligopeptide-binding protein, anoligopeptide ABC transporter/permease protein, a zinc ABCtransporter/zinc-binding protein, a sugar ABC transporter, a phosphatetransporter, a ZIP zinc transporter, a drug resistance transporter ofthe EmrB/QacA family, a sulfate transporter, a proton-dependentoligopeptide transporter, a magnesium transporter, a formate/nitritetransporter, a spermidine/putrescine ABC transporter, aNa/Pi-cotransporter, a sugar phosphate transporter, a glutamine ABCtransporter, a major facilitator family transporter, a glycinebetaine/L-proline ABC transporter, a molybdenum ABC transporter, atechoic acid ABC transporter, a cobalt ABC transporter, an ammoniumtransporter, an amino acid ABC transporter, a cell division ABCtransporter, a manganese ABC transporter, an iron compound ABCtransporter, a maltose/maltodextrin ABC transporter, a drug resistancetransporter of the Bcr/CflA family, and a subunit of one of the aboveproteins.

Other attenuated bacteria and Listeria strains can be deficient in anendogenous metabolic enzyme that metabolizes an amino acid that is usedfor a bacterial growth process, a replication process, cell wallsynthesis, protein synthesis, metabolism of a fatty acid, or for anyother growth or replication process. Likewise, an attenuated strain canbe deficient in an endogenous metabolic enzyme that can catalyze theformation of an amino acid used in cell wall synthesis, can catalyze thesynthesis of an amino acid used in cell wall synthesis, or can beinvolved in synthesis of an amino acid used in cell wall synthesis.Alternatively, the amino acid can be used in cell wall biogenesis.Alternatively, the metabolic enzyme is a synthetic enzyme for D-glutamicacid, a cell wall component.

Other attenuated Listeria strains can be deficient in metabolic enzymesencoded by a D-glutamic acid synthesis gene, dga, an alr (alanineracemase) gene, or any other enzymes that are involved in alaninesynthesis. Yet other examples of metabolic enzymes for which theListeria strain can be deficient include enzymes encoded by serC (aphosphoserine aminotransferase), asd (aspartate betasemialdehydedehydrogenase; involved in synthesis of the cell wall constituentdiaminopimelic acid), the gene encoding gsaB—glutamate-1-semialdehydeaminotransferase (catalyzes the formation of 5-aminolevulinate from(S)-4-amino-5-oxopentanoate), hemL (catalyzes the formation of5-aminolevulinate from (S)-4-amino-5-oxopentanoate), aspB (an aspartateaminotransferase that catalyzes the formation of oxalozcetate andL-glutamate from L-aspartate and 2-oxoglutarate), argF-1 (involved inarginine biosynthesis), aroE (involved in amino acid biosynthesis), aroB(involved in 3-dehydroquinate biosynthesis), aroD (involved in aminoacid biosynthesis), aroC (involved in amino acid biosynthesis), hisB(involved in histidine biosynthesis), hisD (involved in histidinebiosynthesis), hisG (involved in histidine biosynthesis), metX (involvedin methionine biosynthesis), proB (involved in proline biosynthesis),argR (involved in arginine biosynthesis), argJ (involved in argininebiosynthesis), thil (involved in thiamine biosynthesis), LMOf2365_1652(involved in tryptophan biosynthesis), aroA (involved in tryptophanbiosynthesis), ilvD (involved in valine and isoleucine biosynthesis),ilvC (involved in valine and isoleucine biosynthesis), leuA (involved inleucine biosynthesis), dapF (involved in lysine biosynthesis), and thrB(involved in threonine biosynthesis) (all GenBank Accession No.NC_002973).

An attenuated Listeria strain can be generated by mutation of othermetabolic enzymes, such as a tRNA synthetase. For example, the metabolicenzyme can be encoded by the trpS gene, encoding tryptophanyl-tRNAsynthetase. For example, the host strain bacteria can be Δ(trpS aroA),and both markers can be contained in an integration vector.

Other examples of metabolic enzymes that can be mutated to generate anattenuated Listeria strain include an enzyme encoded by murE (involvedin synthesis of diaminopimelic acid; GenBank Accession No: NC_003485),LMOf2365_2494 (involved in teichoic acid biosynthesis), WecE(Lipopolysaccharide biosynthesis protein rffA; GenBank Accession No:AE014075.1), or amiA (an N-acetylmuramoyl-L-alanine amidase). Yet otherexamples of metabolic enzymes include aspartate aminotransferase,histidinol-phosphate aminotransferase (GenBank Accession No. NP_466347),or the cell wall teichoic acid glycosylation protein GtcA.

Other examples of metabolic enzymes that can be mutated to generate anattenuated Listeria strain include a synthetic enzyme for apeptidoglycan component or precursor. The component can be, for example,UDP-N-acetylmuramylpentapeptide, UDP-N-acetylglucosamine,MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol,GlcNAc-p-(1,4)-MurNAc-(pentapeptide)-pyrophosphorylundecaprenol, or anyother peptidoglycan component or precursor.

Yet other examples of metabolic enzymes that can be mutated to generatean attenuated Listeria strain include metabolic enzymes encoded by murG,murD, murA-1, or murA-2 (all set forth in GenBank Accession No.NC_002973). Alternatively, the metabolic enzyme can be any othersynthetic enzyme for a peptidoglycan component or precursor. Themetabolic enzyme can also be a trans-glycosylase, a trans-peptidase, acarboxy-peptidase, any other class of metabolic enzyme, or any othermetabolic enzyme. For example, the metabolic enzyme can be any otherListeria metabolic enzyme or any other Listeria monocytogenes metabolicenzyme.

Other bacteria strains can be attenuated as described above for Listeriaby mutating the corresponding orthologous genes in the other bacteriastrains.

(2) Methods of Complementing Attenuated Bacteria and Listeria Strains

The attenuated bacteria or Listeria strains disclosed herein can furthercomprise a nucleic acid comprising a complementing gene or encoding ametabolic enzyme that complements an attenuating mutation (e.g.,complements the auxotrophy of the auxotrophic Listeria strain). Forexample, a nucleic acid having a first open reading frame encoding afusion polypeptide as disclosed herein can further comprise a secondopen reading frame comprising the complementing gene or encoding thecomplementing metabolic enzyme. Alternatively, a first nucleic acid canencode the fusion polypeptide and a separate second nucleic acid cancomprise the complementing gene or encode the complementing metabolicenzyme.

The complementing gene can be extrachromosomal or can be integrated intothe bacteria or Listeria genome. For example, the auxotrophic Listeriastrain can comprise an episomal plasmid comprising a nucleic acidencoding a metabolic enzyme. Such plasmids will be contained in theListeria in an episomal or extrachromosomal fashion. Alternatively, theauxotrophic Listeria strain can comprise an integrative plasmid (i.e.,integration vector) comprising a nucleic acid encoding a metabolicenzyme. Such integrative plasmids can be used for integration into aListeria chromosome. In some embodiments, the episomal plasmid or theintegrative plasmid lacks an antibiotic resistance marker.

The metabolic gene can be used for selection instead of or in additionto an antibiotic resistance gene. As an example, in order to select forauxotrophic bacteria comprising a plasmid encoding a metabolic enzyme ora complementing gene provided herein, transformed auxotrophic bacteriacan be grown in a medium that will select for expression of the geneencoding the metabolic enzyme (e.g., amino acid metabolism gene) or thecomplementing gene. For example, a bacteria auxotrophic for D-glutamicacid synthesis can be transformed with a plasmid comprising a gene forD-glutamic acid synthesis, and the auxotrophic bacteria will grow in theabsence of D-glutamic acid, whereas auxotrophic bacteria that have notbeen transformed with the plasmid, or are not expressing the plasmidencoding a protein for D-glutamic acid synthesis, will not grow.Similarly, a bacterium auxotrophic for D-alanine synthesis will grow inthe absence of D-alanine when transformed and expressing a plasmidcomprising a nucleic acid encoding an amino acid metabolism enzyme forD-alanine synthesis. Such methods for making appropriate mediacomprising or lacking necessary growth factors, supplements, aminoacids, vitamins, antibiotics, and the like are well-known and areavailable commercially.

Once the auxotrophic bacteria comprising the plasmid encoding ametabolic enzyme or a complementing gene provided herein have beenselected in appropriate medium, the bacteria can be propagated in thepresence of a selective pressure. Such propagation can comprise growingthe bacteria in media without the auxotrophic factor. The presence ofthe plasmid expressing the metabolic enzyme or the complementing gene inthe auxotrophic bacteria ensures that the plasmid will replicate alongwith the bacteria, thus continually selecting for bacteria harboring theplasmid. Production of the bacteria or Listeria strain can be readilyscaled up by adjusting the volume of the medium in which the auxotrophicbacteria comprising the plasmid are growing.

In one specific example, the attenuated strain is a strain having adeletion of or an inactivating mutation in dal and dat (e.g., Listeriamonocytogenes (Lm) dal(−)dat(−) (Lmdd) or Lm dal(−)dat(−)ΔactA (LmddA)),and the complementing gene encodes an alanine racemase enzyme (e.g.,encoded by dal gene) or a D-amino acid aminotransferase enzyme (e.g.,encoded by dat gene). An exemplary alanine racemase protein can have thesequence set forth in SEQ ID NO: 76 (encoded by SEQ ID NO: 78; GenBankAccession No: AF038438) or can be a homologue, variant, isoform, analog,fragment, fragment of a homologue, fragment of a variant, fragment of ananalog, or fragment of an isoform of SEQ ID NO: 76. The alanine racemaseprotein can also be any other Listeria alanine racemase protein.Alternatively, the alanine racemase protein can be any othergram-positive alanine racemase protein or any other alanine racemaseprotein. An exemplary D-amino acid aminotransferase protein can have thesequence set forth in SEQ ID NO: 77 (encoded by SEQ ID NO: 79; GenBankAccession No: AF038439) or can be a homologue, variant, isoform, analog,fragment, fragment of a homologue, fragment of a variant, fragment of ananalog, or fragment of an isoform of SEQ ID NO: 77. The D-amino acidaminotransferase protein can also be any other Listeria D-amino acidaminotransferase protein. Alternatively, the D-amino acidaminotransferase protein can be any other gram-positive D-amino acidaminotransferase protein or any other D-amino acid aminotransferaseprotein.

In another specific example, the attenuated strain is a strain having adeletion of or an inactivating mutation in prfA (e.g., Lm prfA(−)), andthe complementing gene encodes a PrfA protein. For example, thecomplementing gene can encode a mutant PrfA (D133V) protein thatrestores partial PrfA function. An example of a wild type PrfA proteinis set forth in SEQ ID NO: 80 (encoded by nucleic acid set forth in SEQID NO: 81), and an example of a D133V mutant PrfA protein is set forthin SEQ ID NO: 82 (encoded by nucleic acid set forth in SEQ ID NO: 83).The complementing PrfA protein can be a homologue, variant, isoform,analog, fragment, fragment of a homologue, fragment of a variant,fragment of an analog, or fragment of an isoform of SEQ ID NO: 80 or 82.The PrfA protein can also be any other Listeria PrfA protein.Alternatively, the PrfA protein can be any other gram-positive PrfAprotein or any other PrfA protein.

In another example, the bacteria strain or Listeria strain can comprisea deletion of or an inactivating mutation in an actA gene, and thecomplementing gene can comprise an actA gene to complement the mutationand restore function to the Listeria strain.

Other auxotroph strains and complementation systems can also be adoptedfor the use with the methods and compositions provided herein.

IV. Recombinant Fusion Polypeptides

The recombinant fusion polypeptides in the recombinant bacteria orListeria strains disclosed herein can be in any form. Some such fusionpolypeptides can comprise a PEST-containing peptide fused to one or moredisease-associated antigenic peptides. Other such recombinant fusionpolypeptides can comprise one or more disease-associated antigenicpeptides, and wherein the fusion polypeptide does not comprise aPEST-containing peptide.

Another example of a recombinant fusion polypeptides comprises fromN-terminal end to C-terminal end a bacterial secretion sequence, aubiquitin (Ub) protein, and one or more disease-associated antigenicpeptides (i.e., in tandem, such as Ub-peptide1-peptide2). Alternatively,if two or more disease-associated antigenic peptides are used, acombination of separate fusion polypeptides can be used in which eachantigenic peptide is fused to its own secretion sequence and Ub protein(e.g., Ub1-peptide1; Ub2-peptide2).

Nucleic acids (termed minigene constructs) encoding such recombinantfusion polypeptides are also disclosed. Such minigene nucleic acidconstructs can further comprise two or more open reading frames linkedby a Shine-Dalgarno ribosome binding site nucleic acid sequence betweeneach open reading frame. For example, a minigene nucleic acid constructcan further comprise two to four open reading frames linked by aShine-Dalgarno ribosome binding site nucleic acid sequence between eachopen reading frame. Each open reading frame can encode a differentpolypeptide. In some nucleic acid constructs, the codon encoding thecarboxy terminus of the fusion polypeptide is followed by two stopcodons to ensure termination of protein synthesis.

The bacterial signal sequence can be a Listerial signal sequence, suchas an Hly or an ActA signal sequence, or any other known signalsequence. In other cases, the signal sequence can be an LLO signalsequence. An exemplary LLO signal sequence is set forth in SEQ ID NO:97. The signal sequence can be bacterial, can be native to a hostbacterium (e.g., Listeria monocytogenes, such as a secA1 signalpeptide), or can be foreign to a host bacterium. Specific examples ofsignal peptides include an Usp45 signal peptide from Lactococcus lactis,a Protective Antigen signal peptide from Bacillus anthracis, a secA2signal peptide such the p60 signal peptide from Listeria monocytogenes,and a Tat signal peptide such as a B. subtilis Tat signal peptide (e.g.,PhoD). In specific examples, the secretion signal sequence is from aListeria protein, such as an ActA₃₀₀ secretion signal or an ActA₁₀₀secretion signal. An exemplary ActA signal sequence is set forth in SEQID NO: 98.

The ubiquitin can be, for example, a full-length protein. The ubiquitinexpressed from the nucleic acid construct provided herein can be cleavedat the carboxy terminus from the rest of the recombinant fusionpolypeptide expressed from the nucleic acid construct through the actionof hydrolases upon entry to the host cell cytosol. This liberates theamino terminus of the fusion polypeptide, producing a peptide in thehost cell cytosol.

Selection of, variations of, and arrangement of antigenic peptideswithin a fusion polypeptide are discussed in detail elsewhere herein,and examples of disease-associated antigenic peptides are discussed inmore detail elsewhere herein.

The recombinant fusion polypeptides can comprise one or more tags. Forexample, the recombinant fusion polypeptides can comprise one or morepeptide tags N-terminal and/or C-terminal to one or more antigenicpeptides. A tag can be fused directly to an antigenic peptide or linkedto an antigenic peptide via a linker (examples of which are disclosedelsewhere herein). Examples of tags include the following: FLAG tag;2×FLAG tag; 3×FLAG tag; His tag, 6×His tag; and SIINFEKL tag. Anexemplary SIINFEKL tag is set forth in SEQ ID NO: 16 (encoded by any oneof the nucleic acids set forth in SEQ ID NOS: 1-15). An exemplary 3×FLAGtag is set forth in SEQ ID NO: 32 (encoded by any one of the nucleicacids set forth in SEQ ID NOS: 17-31). An exemplary variant 3×FLAG tagis set forth in SEQ ID NO: 99. Two or more tags can be used together,such as a 2×FLAG tag and a SIINFEKL tag, a 3×FLAG tag and a SIINFEKLtag, or a 6×His tag and a SIINFEKL tag. If two or more tags are used,they can be located anywhere within the recombinant fusion polypeptideand in any order. For example, the two tags can be at the C-terminus ofthe recombinant fusion polypeptide, the two tags can be at theN-terminus of the recombinant fusion polypeptide, the two tags can belocated internally within the recombinant fusion polypeptide, one tagcan be at the C-terminus and one tag at the N-terminus of therecombinant fusion polypeptide, one tag can be at the C-terminus and oneinternally within the recombinant fusion polypeptide, or one tag can beat the N-terminus and one internally within the recombinant fusionpolypeptide. Other tags include chitin binding protein (CBP), maltosebinding protein (MBP), glutathione-S-transferase (GST), thioredoxin(TRX), and poly(NANP). Particular recombinant fusion polypeptidescomprise a C-terminal SIINFEKL tag. Such tags can allow for easydetection of the recombinant fusion protein, confirmation of secretionof the recombinant fusion protein, or for following the immunogenicityof the secreted fusion polypeptide by following immune responses tothese “tag” sequence peptides. Such immune response can be monitoredusing a number of reagents including, for example, monoclonal antibodiesand DNA or RNA probes specific for these tags.

The recombinant fusion polypeptides disclosed herein can be expressed byrecombinant Listeria strains or can be expressed and isolated from othervectors and cell systems used for protein expression and isolation.Recombinant Listeria strains comprising expressing such antigenicpeptides can be used, for example in immunogenic compositions comprisingsuch recombinant Listeria and in vaccines comprising the recombinantListeria strain and an adjuvant. Expression of one or more antigenicpeptides as a fusion polypeptides with a nonhemolytic truncated form ofLLO, ActA, or a PEST-like sequence in host cell systems in Listeriastrains and host cell systems other than Listeria can result in enhancedimmunogenicity of the antigenic peptides.

Nucleic acids encoding such recombinant fusion polypeptides are alsodisclosed. The nucleic acid can be in any form. The nucleic acid cancomprise or consist of DNA or RNA, and can be single-stranded ordouble-stranded. The nucleic acid can be in the form of a plasmid, suchas an episomal plasmid, a multicopy episomal plasmid, or an integrativeplasmid. Alternatively, the nucleic acid can be in the form of a viralvector, a phage vector, or in a bacterial artificial chromosome. Suchnucleic acids can have one open reading frame or can have two or moreopen reading frames (e.g., an open reading frame encoding therecombinant fusion polypeptide and a second open reading frame encodinga metabolic enzyme). In one example, such nucleic acids can comprise twoor more open reading frames linked by a Shine-Dalgarno ribosome bindingsite nucleic acid sequence between each open reading frame. For example,a nucleic acid can comprise two to four open reading frames linked by aShine-Dalgarno ribosome binding site nucleic acid sequence between eachopen reading frame. Each open reading frame can encode a differentpolypeptide. In some nucleic acids, the codon encoding the carboxyterminus of the fusion polypeptide is followed by two stop codons toensure termination of protein synthesis.

A. Antigenic Peptides

Disease-associated peptides include peptides from proteins that areexpressed in a particular disease. For example, such peptides may befrom proteins that are expressed in a disease tissue but not in acorresponding normal tissue, or that are expressed at abnormally highlevels in a disease tissue. The term “disease” as used herein isintended to be generally synonymous, and is used interchangeably with,the terms “disorder” and “condition” (as in medical condition), in thatall reflect an abnormal condition of the human or animal body or of oneof its parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life. Examples ofdisease-associated antigenic peptides can include Human Papilloma Virus(HPV) E7 or E6, a Prostate Specific Antigen (PSA), a chimeric Her2antigen, Her2/neu chimeric antigen. Another example of adisease-associated antigenic peptide is a WT1 antigenic peptide. TheHuman Papilloma Virus can be HPV 16 or HPV 18. The antigenic peptide canalso include HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 antigens operablylinked in tandem or HPV16 antigenic peptide operably linked in tandem toan HPV antigenic peptide.

The fusion polypeptide can include a single antigenic peptide or canincludes two or more antigenic peptides. Each antigenic peptide can beof any length sufficient to induce an immune response, and eachantigenic peptide can be the same length or the antigenic peptides canhave different lengths. For example, an antigenic peptide disclosedherein can be 5-100, 15-50, or 21-27 amino acids in length, or 15-100,15-95, 15-90, 15-85, 15-80, 15-75, 15-70, 15-65, 15-60, 15-55, 15-50,15-45, 15-40, 15-35, 15-30, 20-100, 20-95, 20-90, 20-85, 20-80, 20-75,20-70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 11-21,15-21, 21-31, 31-41, 41-51, 51-61, 61-71, 71-81, 81-91, 91-101, 101-121,121-141, 141-161, 161-181, 181-201, 8-27, 10-30, 10-40, 15-30, 15-40,15-25, 1-10, 10-20, 20-30, 30-40, 1-100, 5-75, 5-50, 5-40, 5-30, 5-20,5-15, 5-10, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 8-11, or 11-16amino acids in length. For example, an antigenic peptide can be at least15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids in length. Somespecific examples of antigenic peptides are 21 or 27 amino acids inlength. Other antigenic peptides can be full-length proteins orfragments thereof.

As one example, an antigenic peptide can comprise a neoepitope. Theseneoepitopes can be, for example, patient-specific (i.e.,subject-specific) cancer mutations. Antigenic peptides comprisingneoepitopes can be generated in a process for creating a personalizedimmunotherapy comprising comparing nucleic acids extracted from a cancersample from a subject to nucleic acids extracted from a normal orhealthy reference sample in order to identify somatic mutations orsequence differences present in the cancer sample compared with thenormal or healthy sample. For examples, these mutations or sequencedifferences can be somatic, nonsynonymous missense mutations, or somaticframeshift mutations, and can encode an expressed amino acid sequence. Apeptide expressing such somatic mutations or sequence differences can bereferred to as a “neoepitope.” A cancer-specific neoepitope may refer toan epitope that is not present in a reference sample (such as a normalnon-cancerous or germline cell or tissue) but is found in a cancersample. This includes, for example, situations in which in a normalnon-cancerous or germline cell a corresponding epitope is found, but dueto one or more mutations in a cancer cell, the sequence of the epitopeis changed so as to result in the neoepitope. A neoepitope can comprisea mutated epitope, and can comprise non-mutated sequence on either orboth sides of the mutation.

As another example, antigenic peptides can comprise recurrent cancermutations. Each antigenic peptide can comprise a single recurrent cancermutation or can comprise two or more recurrent cancer mutations (e.g.,two recurrent cancer mutations). For example, an antigenic peptide cancomprise more than one recurrent cancer mutation (e.g., 2 or 3 recurrentcancer mutations) because of the close proximity of the mutated residuesto each other in the cancer-associated protein. The recurrent cancermutations can be any type of mutation (e.g., somatic missense mutationor frameshift mutation). For example, a recombinant fusion polypeptidedisclosed herein can comprise a PEST-containing peptide fused to two ormore antigenic peptides (i.e., in tandem, such asPEST-peptide1-peptide2) or can comprise two or more antigenic peptidesnot fused to a PEST-containing peptide, wherein each antigenic peptidecomprises a single, recurrent cancer mutation (i.e., a single, recurrentchange in the amino acid sequence of a protein, or a sequence encoded bya single, different, nonsynonymous, recurrent cancer mutation in agene), and wherein at least two of the antigenic peptides comprisedifferent recurrent cancer mutations and are fragments of the samecancer-associated protein. Alternatively, each of the antigenic peptidescan comprise a different recurrent cancer mutation from a differentcancer-associated protein. Alternatively, a combination of separatefusion polypeptides can be used in which each antigenic peptide is fused(or is not fused) to its own PEST-containing peptide (e.g.,PEST1-peptide1; PEST2-peptide2). Optionally, some or all of thefragments are non-contiguous fragments of the same cancer-associatedprotein. Non-contiguous fragments are fragments that do not occursequentially in a protein sequence (e.g., the first fragment consists ofresidues 10-30, and the second fragment consists of residues 100-120; orthe first fragment consists of residues 10-30, and the second fragmentconsists of residues 20-40). Optionally, each of the antigenic peptidescomprises a different recurrent cancer mutation from a single type ofcancer.

Recurrent cancer mutations can be from cancer-associated proteins. Theterm “cancer-associated protein” includes proteins having mutations thatoccur in multiple types of cancer, that occur in multiple subjectshaving a particular type of cancer, or that are correlated with theoccurrence or progression of one or more types of cancer. For example, acancer-associated protein can be an oncogenic protein (i.e., a proteinwith activity that can contribute to cancer progression, such asproteins that regulate cell growth), or it can be a tumor-suppressorprotein (i.e., a protein that typically acts to alleviate the potentialfor cancer formation, such as through negative regulation of the cellcycle or by promoting apoptosis). In some embodiments, acancer-associated protein has a “mutational hotspot.” A mutationalhotspot is an amino acid position in a protein-coding gene that ismutated (such as by somatic substitutions rather than other somaticabnormalities, such as translocations, amplifications, and deletions)more frequently than would be expected in the absence of selection. Suchhotspot mutations can occur across multiple types of cancer and/or canbe shared among multiple cancer patients. Mutational hotspots indicateselective pressure across a population of tumor samples. Tumor genomescontain recurrent cancer mutations that “drive” tumorigenesis byaffecting genes (i.e., tumor driver genes) that confer selective growthadvantages to the tumor cells upon alteration. Such tumor driver genescan be identified, for example, by identifying genes that are mutatedmore frequently than expected from the background mutation rate (i.e.,recurrence); by identifying genes that exhibit other signals of positiveselection across tumor samples (e.g., a high rate of non-silentmutations compared to silent mutations, or a bias towards theaccumulation of functional mutations); by exploiting the tendency tosustain mutations in certain regions of the protein sequence based onthe knowledge that whereas inactivating mutations are distributed alongthe sequence of the protein, gain-of-function mutations tend to occurspecifically in particular residues or domains; or by exploiting theoverrepresentation of mutations in specific functional residues, such asphosphorylation sites. Many of these mutations frequently occur in thefunctional regions of biologically active proteins (for example, kinasedomains or binding domains) or interrupt active sites (for example,phosphorylation sites) resulting in loss-of-function or gain-of-functionmutations, or they can occur in such a way that the three-dimensionalstructure and/or charge balance of the protein is perturbed sufficientlyto interfere with normal function. Genomic analysis of large numbers oftumors reveals that mutations often occur at a limited number of aminoacid positions. Therefore, a majority of the common mutations can berepresented by a relatively small number of potential tumor-associatedantigens or T cell epitopes.

A “recurrent cancer mutation” is a change in the amino acid sequence ofa protein that occurs in multiple types of cancer and/or in multiplesubjects having a particular types of cancer. Such mutations associatedwith a cancer can result in tumor-associated antigens that are notnormally present in corresponding healthy tissue.

Tumor-driver genes and cancer-associated proteins having commonmutations that occur across multiple cancers or among multiple cancerpatients are known, and sequencing data across multiple tumor samplesand multiple tumor types exists. See, e.g., Chang et al. (2016) NatBiotechnol 34(2):155-163; Tamborero et al. (2013) Sci Rep 3:2650, eachof which is herein incorporated by reference in its entirety.

As another example, an antigenic peptide can be a heteroclitic antigenicpeptide. For example, a heteroclitic antigenic peptide can be a fragmentof a cancer-associated protein (i.e., a contiguous sequence of aminoacids from a cancer-associated protein) comprising a heterocliticmutation. A heteroclitic antigenic peptide can comprise a singleheteroclitic mutation or can comprise two or more heteroclitic mutations(e.g., two heteroclitic mutations). The term “heteroclitic” refers to apeptide that generates an immune response that recognizes the nativepeptide from which the heteroclitic peptide was derived (e.g., thepeptide not containing the anchor residue mutations).

Some recombinant fusion polypeptides disclosed herein can comprise anycombination of antigenic peptides comprising recurrent cancer mutations,antigenic peptides (e.g., from cancer-associated proteins) comprisingheteroclitic mutations, and antigenic peptides (e.g., fromcancer-associated proteins) expressed from minigene constructs (i.e.,antigenic peptides such as a heteroclitic antigenic peptide fused toubiquitin). For example, such a recombinant fusion polypeptide cancomprise a PEST-containing peptide fused to two or more antigenicpeptides, wherein at least one antigenic peptide is from acancer-associated protein and comprises a recurrent cancer mutation, andat least one antigenic peptide is from a cancer-associated protein andcomprises a heteroclitic mutation. Optionally, the PEST-containingpeptide comprises a bacterial secretion signal sequence, and the fusionpolypeptide further comprises a ubiquitin protein fused to acarboxy-terminal antigenic peptide, wherein the PEST-containing peptide,the two or more antigenic peptides, the ubiquitin, and thecarboxy-terminal antigenic peptide are arranged in tandem from theamino-terminal end to the carboxy-terminal end of the fusionpolypeptide.

Each antigenic peptide can also be hydrophilic or can score up to orbelow a certain hydropathy threshold, which can be predictive ofsecretability in Listeria monocytogenes or another bacteria of interest.For example, antigenic peptides can be scored by a Kyte and Doolittlehydropathy index 21 amino acid window, and all scoring above a cutoff(around 1.6) can be excluded as they are unlikely to be secretable byListeria monocytogenes. Likewise, the combination of antigenic peptidesor the fusion polypeptide can be hydrophilic or can score up to or belowa certain hydropathy threshold, which can be predictive of secretabilityin Listeria monocytogenes or another bacteria of interest.

The antigenic peptides can be linked together in any manner. Forexample, the antigenic peptides can be fused directly to each other withno intervening sequence. Alternatively, the antigenic peptides can belinked to each other indirectly via one or more linkers, such as peptidelinkers. In some cases, some pairs of adjacent antigenic peptides can befused directly to each other, and other pairs of antigenic peptides canbe linked to each other indirectly via one or more linkers. The samelinker can be used between each pair of adjacent antigenic peptides, orany number of different linkers can be used between different pairs ofadjacent antigenic peptides. In addition, one linker can be used betweena pair of adjacent antigenic peptides, or multiple linkers can be usedbetween a pair of adjacent antigenic peptides.

Any suitable sequence can be used for a peptide linker. As an example, alinker sequence may be, for example, from 1 to about 50 amino acids inlength. Some linkers may be hydrophilic. The linkers can serve varyingpurposes. For example, the linkers can serve to increase bacterialsecretion, to facilitate antigen processing, to increase flexibility ofthe fusion polypeptide, to increase rigidity of the fusion polypeptide,or any other purpose. In some cases, different amino acid linkersequences are distributed between the antigenic peptides or differentnucleic acids encoding the same amino acid linker sequence aredistributed between the antigenic peptides (e.g., SEQ ID NOS: 84-94) inorder to minimize repeats. This can also serve to reduce secondarystructures, thereby allowing efficient transcription, translation,secretion, maintenance, or stabilization of the nucleic acid (e.g.,plasmid) encoding the fusion polypeptide within a Lm recombinant vectorstrain population. Other suitable peptide linker sequences may bechosen, for example, based on one or more of the following factors: (1)their ability to adopt a flexible extended conformation; (2) theirinability to adopt a secondary structure that could interact withfunctional epitopes on the antigenic peptides; and (3) the lack ofhydrophobic or charged residues that might react with the functionalepitopes. For example, peptide linker sequences may contain Gly, Asn andSer residues. Other near neutral amino acids, such as Thr and Ala mayalso be used in the linker sequence. Amino acid sequences which may beusefully employed as linkers include those disclosed in Maratea et al.(1985) Gene 40:39-46; Murphy et al. (1986) Proc Natl Acad Sci USA83:8258-8262; U.S. Pat. Nos. 4,935,233; and 4,751,180, each of which isherein incorporated by reference in its entirety for all purposes.Specific examples of linkers include those in Table 2 (each of which canbe used by itself as a linker, in a linker comprising repeats of thesequence, or in a linker further comprising one or more of the othersequences in the table), although others can also be envisioned (see,e.g., Reddy Chichili et al. (2013) Protein Science 22:153-167, hereinincorporated by reference in its entirety for all purposes). Unlessspecified, “n” represents an undetermined number of repeats in thelisted linker.

TABLE 2 Linkers. Hypothetical Peptide Linker Example SEQ ID NO: Purpose(GAS)_(n) GASGAS 33 Flexibility (GSA)_(n) GSAGSA 34 Flexibility (G)_(n);n = 4-8 GGGG 35 Flexibility (GGGGS)_(n); n = 1-3 GGGGS 36 FlexibilityVGKGGSGG VGKGGSGG 37 Flexibility (PAPAP)_(n) PAPAP 38 Rigidity(EAAAK)_(n); n = 1-3 EAAAK 39 Rigidity (AYL)_(n) AYLAYL 40 AntigenProcessing (LRA)_(n) LRALRA 41 Antigen Processing (RLRA)_(n) RLRA 42Antigen Processing

B. PEST-Containing Peptides

The recombinant fusion proteins disclosed herein comprise aPEST-containing peptide. The PEST-containing peptide may at the aminoterminal (N-terminal) end of the fusion polypeptide (i.e., N-terminal tothe antigenic peptides), may be at the carboxy terminal (C-terminal) endof the fusion polypeptide (i.e., C-terminal to the antigenic peptides),or may be embedded within the antigenic peptides. In some recombinantListeria strains and methods, a PEST containing peptide is not part ofand is separate from the fusion polypeptide. Fusion of an antigenicpeptides to a PEST-like sequence, such as an LLO peptide, can enhancethe immunogenicity of the antigenic peptides and can increasecell-mediated and antitumor immune responses (i.e., increasecell-mediated and anti-tumor immunity). See, e.g., Singh et al. (2005) JImmunol 175(6):3663-3673, herein incorporated by reference in itsentirety for all purposes.

A PEST-containing peptide is one that comprises a PEST sequence or aPEST-like sequence. PEST sequences in eukaryotic proteins have long beenidentified. For example, proteins containing amino acid sequences thatare rich in prolines (P), glutamic acids (E), serines (S) and threonines(T) (PEST), generally, but not always, flanked by clusters containingseveral positively charged amino acids, have rapid intracellularhalf-lives (Rogers et al. (1986) Science 234:364-369, hereinincorporated by reference in its entirety for all purposes). Further, ithas been reported that these sequences target the protein to theubiquitin-proteasome pathway for degradation (Rechsteiner and Rogers(1996) Trends Biochem. Sci. 21:267-271, herein incorporated by referencein its entirety for all purposes). This pathway is also used byeukaryotic cells to generate immunogenic peptides that bind to MHC classI and it has been hypothesized that PEST sequences are abundant amongeukaryotic proteins that give rise to immunogenic peptides (Realini etal. (1994) FEBS Lett. 348:109-113, herein incorporated by reference inits entirety for all purposes). Prokaryotic proteins do not normallycontain PEST sequences because they do not have this enzymatic pathway.However, a PEST-like sequence rich in the amino acids proline (P),glutamic acid (E), serine (S) and threonine (T) has been reported at theamino terminus of LLO and has been reported to be essential for L.monocytogenes pathogenicity (Decatur and Portnoy (2000) Science290:992-995, herein incorporated by reference in its entirety for allpurposes). The presence of this PEST-like sequence in LLO targets theprotein for destruction by proteolytic machinery of the host cell sothat once the LLO has served its function and facilitated the escape ofL. monocytogenes from the phagosomal or phagolysosomal vacuole, it isdestroyed before it can damage the cells.

Identification of PEST and PEST-like sequences is well-known and isdescribed, for example, in Rogers et al. (1986) Science234(4774):364-378 and in Rechsteiner and Rogers (1996) Trends Biochem.Sci. 21:267-271, each of which is herein incorporated by reference inits entirety for all purposes. A PEST or PEST-like sequence can beidentified using the PEST-find program. For example, a PEST-likesequence can be a region rich in proline (P), glutamic acid (E), serine(S), and threonine (T) residues. Optionally, the PEST-like sequence canbe flanked by one or more clusters containing several positively chargedamino acids. For example, a PEST-like sequence can be defined as ahydrophilic stretch of at least 12 amino acids in length with a highlocal concentration of proline (P), aspartate (D), glutamate (E), serine(S), and/or threonine (T) residues. In some cases, a PEST-like sequencecontains no positively charged amino acids, namely arginine (R),histidine (H), and lysine (K). Some PEST-like sequences can contain oneor more internal phosphorylation sites, and phosphorylation at thesesites precedes protein degradation.

In one example, the PEST-like sequence fits an algorithm disclosed inRogers et al. In another example, the PEST-like sequence fits analgorithm disclosed in Rechsteiner and Rogers. PEST-like sequences canalso be identified by an initial scan for positively charged amino acidsR, H, and K within the specified protein sequence. All amino acidsbetween the positively charged flanks are counted, and only those motifscontaining a number of amino acids equal to or higher than thewindow-size parameter are considered further. Optionally, a PEST-likesequence must contain at least one P, at least one D or E, and at leastone S or T.

The quality of a PEST motif can be refined by means of a scoringparameter based on the local enrichment of critical amino acids as wellas the motifs hydrophobicity. Enrichment of D, E, P, S, and T isexpressed in mass percent (w/w) and corrected for one equivalent of D orE, one1 of P, and one of S or T. Calculation of hydrophobicity can alsofollow in principle the method of Kyte and Doolittle (1982) J. Mol.Biol. 157:105, herein incorporated by reference in its entirety for allpurposes. For simplified calculations, Kyte-Doolittle hydropathyindices, which originally ranged from −4.5 for arginine to +4.5 forisoleucine, are converted to positive integers, using the followinglinear transformation, which yielded values from 0 for arginine to 90for isoleucine: Hydropathy index=10*Kyte-Doolittle hydropathy index+45.

A potential PEST motif's hydrophobicity can also be calculated as thesum over the products of mole percent and hydrophobicity index for eachamino acid species. The desired PEST score is obtained as combination oflocal enrichment term and hydrophobicity term as expressed by thefollowing equation: PEST score=0.55*DEPST−0.5*hydrophobicity index.

Thus, a PEST-containing peptide can refer to a peptide having a score ofat least +5 using the above algorithm. Alternatively, it can refer to apeptide having a score of at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least32, at least 35, at least 38, at least 40, or at least 45.

Any other known available methods or algorithms can also be used toidentify PEST-like sequences. See, e.g., the CaSPredictor(Garay-Malpartida et al. (2005) Bioinformatics 21 Suppl 1:i169-76,herein incorporated by reference in its entirety for all purposes).Another method that can be used is the following: a PEST index iscalculated for each stretch of appropriate length (e.g. a 30-35 aminoacid stretch) by assigning a value of one to the amino acids Ser, Thr,Pro, Glu, Asp, Asn, or Gln. The coefficient value (CV) for each of thePEST residues is one and the CV for each of the other AA (non-PEST) iszero.

Examples of PEST-like amino acid sequences are those set forth in SEQ IDNOS: 43-51. One example of a PEST-like sequence isKENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 43). Another example of aPEST-like sequence is KENSISSMAPPASPPASPK (SEQ ID NO: 44). However, anyPEST or PEST-like amino acid sequence can be used. PEST sequencepeptides are known and are described, for example, in U.S. Pat. Nos.7,635,479; 7,665,238; and US 2014/0186387, each of which is hereinincorporated by reference in its entirety for all purposes.

The PEST-like sequence can be from a Listeria species, such as fromListeria monocytogenes. For example, the Listeria monocytogenes ActAprotein contains at least four such sequences (SEQ ID NOS: 45-48), anyof which are suitable for use in the compositions and methods disclosedherein. Other similar PEST-like sequences include SEQ ID NOS: 52-54.Streptolysin O proteins from Streptococcus sp. also contain a PESTsequence. For example, Streptococcus pyogenes streptolysin O comprisesthe PEST sequence KQNTASTETTTTNEQPK (SEQ ID NO: 49) at amino acids 35-51and Streptococcus equisimilis streptolysin O comprises the PEST-likesequence KQNTANTETTTTNEQPK (SEQ ID NO: 50) at amino acids 38-54. Anotherexample of a PEST-like sequence is from Listeria seeligeri cytolysin,encoded by the lso gene: RSEVTISPAETPESPPATP (e.g., SEQ ID NO: 51).

Alternatively, the PEST-like sequence can be derived from otherprokaryotic organisms. Other prokaryotic organisms wherein PEST-likeamino acid sequences would be expected include, for example, otherListeria species.

(1) Listeriolysin O (LLO)

One example of a PEST-containing peptide that can be utilized in thecompositions and methods disclosed herein is a listeriolysin O (LLO)peptide. An example of an LLO protein is the protein assigned GenBankAccession No. P13128 (SEQ ID NO: 55; nucleic acid sequence is set forthin GenBank Accession No. X15127). SEQ ID NO: 55 is a proproteinincluding a signal sequence. The first 25 amino acids of the proproteinis the signal sequence and is cleaved from LLO when it is secreted bythe bacterium, thereby resulting in the full-length active LLO proteinof 504 amino acids without the signal sequence. An LLO peptide disclosedherein can comprise the signal sequence or can comprise a peptide thatdoes not include the signal sequence. Exemplary LLO proteins that can beused comprise, consist essentially of, or consist of the sequence setforth in SEQ ID NO: 55 or homologues, variants, isoforms, analogs,fragments, fragments of homologues, fragments of variants, fragments ofanalogs, and fragments of isoforms of SEQ ID NO: 55. Any sequence thatencodes a fragment of an LLO protein or a homologue, variant, isoform,analog, fragment of a homologue, fragment of a variant, or fragment ofan analog of an LLO protein can be used. A homologous LLO protein canhave a sequence identity with a reference LLO protein, for example, ofgreater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%,93%, 95%, 96%, 97%, 98%, or 99%.

Another example of an LLO protein is set forth in SEQ ID NO: 56. LLOproteins that can be used can comprise, consist essentially of, orconsist of the sequence set forth in SEQ ID NO: 56 or homologues,variants, isoforms, analogs, fragments, fragments of homologues,fragments of variants, fragments of analogs, and fragments of isoformsof SEQ ID NO: 56.

Another example of an LLO protein is an LLO protein from the Listeriamonocytogenes 10403S strain, as set forth in GenBank Accession No.:ZP_01942330 or EBA21833, or as encoded by the nucleic acid sequence asset forth in GenBank Accession No.: NZ_AARZ01000015 or AARZ01000015.1.Another example of an LLO protein is an LLO protein from the Listeriamonocytogenes 4b F2365 strain (see, e.g., GenBank Accession No.:YP_012823), EGD-e strain (see, e.g., GenBank Accession No.: NP_463733),or any other strain of Listeria monocytogenes. Yet another example of anLLO protein is an LLO protein from Flavobacteriales bacterium HTCC2170(see, e.g., GenBank Accession No.: ZP_01106747 or EAR01433, or encodedby GenBank Accession No.: NZ_AAOC01000003). LLO proteins that can beused can comprise, consist essentially of, or consist of any of theabove LLO proteins or homologues, variants, isoforms, analogs,fragments, fragments of homologues, fragments of variants, fragments ofanalogs, and fragments of isoforms of the above LLO proteins.

Proteins that are homologous to LLO, or homologues, variants, isoforms,analogs, fragments, fragments of homologues, fragments of variants,fragments of analogs, and fragments of isoforms thereof, can also beused. One such example is alveolysin, which can be found, for example,in Paenibacillus alvei (see, e.g., GenBank Accession No.: P23564 orAAA22224, or encoded by GenBank Accession No.: M62709). Other suchhomologous proteins are known.

The LLO peptide can be a full-length LLO protein or a truncated LLOprotein or LLO fragment. Likewise, the LLO peptide can be one thatretains one or more functionalities of a native LLO protein or lacks oneor more functionalities of a native LLO protein. For example, theretained LLO functionality can be allowing a bacteria (e.g., Listeria)to escape from a phagosome or phagolysosome, or enhancing theimmunogenicity of a peptide to which it is fused. The retainedfunctionality can also be hemolytic function or antigenic function.Alternatively, the LLO peptide can be a non-hemolytic LLO. Otherfunctions of LLO are known, as are methods and assays for evaluating LLOfunctionality.

An LLO fragment can be a PEST-like sequence or can comprise a PEST-likesequence. LLO fragments can comprise one or more of an internaldeletion, a truncation from the C-terminal end, and a truncation fromthe N-terminal end. In some cases, an LLO fragment can comprise morethan one internal deletion. Other LLO peptides can be full-length LLOproteins with one or more mutations.

Some LLO proteins or fragments have reduced hemolytic activity relativeto wild type LLO or are non-hemolytic fragments. For example, an LLOprotein can be rendered non-hemolytic by deletion or mutation of theactivation domain at the carboxy terminus, by deletion or mutation ofcysteine 484, or by deletion or mutation at another location.

Other LLO proteins are rendered non-hemolytic by a deletion or mutationof the cholesterol binding domain (CBD) as detailed in U.S. Pat. No.8,771,702, herein incorporated by reference in its entirety for allpurposes. The mutations can comprise, for example, a substitution or adeletion. The entire CBD can be mutated, portions of the CBD can bemutated, or specific residues within the CBD can be mutated. Forexample, the LLO protein can comprise a mutation of one or more ofresidues C484, W491, and W492 (e.g., C484, W491, W492, C484 and W491,C484 and W492, W491 and W492, or all three residues) of SEQ ID NO: 55 orcorresponding residues when optimally aligned with SEQ ID NO: 55 (e.g.,a corresponding cysteine or tryptophan residue). As an example, a mutantLLO protein can be created wherein residues C484, W491, and W492 of LLOare substituted with alanine residues, which will substantially reducehemolytic activity relative to wild type LLO. The mutant LLO proteinwith C484A, W491A, and W492A mutations is termed “mutLLO.”

As another example, a mutant LLO protein can be created with an internaldeletion comprising the cholesterol-binding domain. The sequence of thecholesterol-binding domain of SEQ ID NO: 55 set forth in SEQ ID NO: 74.For example, the internal deletion can be a 1-11 amino acid deletion, an11-50 amino acid deletion, or longer. Likewise, the mutated region canbe 1-11 amino acids, 11-50 amino acids, or longer (e.g., 1-50, 1-11,2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10,3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25, 11-30,11-35, 11-40, 11-50, 11-60, 11-70, 11-80, 11-90, 11-100, 11-150, 15-20,15-25, 15-30, 15-35, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100,15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90,20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or30-150 amino acids). For example, a mutated region consisting ofresidues 470-500, 470-510, or 480-500 of SEQ ID NO: 55 will result in adeleted sequence comprising the CBD (residues 483-493 of SEQ ID NO: 55).However, the mutated region can also be a fragment of the CBD or canoverlap with a portion of the CBD. For example, the mutated region canconsist of residues 470-490, 480-488, 485-490, 486-488, 490-500, or486-510 of SEQ ID NO: 55. For example, a fragment of the CBD (residues484-492) can be replaced with a heterologous sequence, which willsubstantially reduce hemolytic activity relative to wild type LLO. Forexample, the CBD (ECTGLAWEWWR; SEQ ID NO: 74) can be replaced with a CTLepitope from the antigen NY-ESO-1 (ESLLMWITQCR; SEQ ID NO: 75), whichcontains the HLA-A2 restricted epitope 157-165 from NY-ESO-1. Theresulting LLO is termed “ctLLO.”

In some mutated LLO proteins, the mutated region can be replaced by aheterologous sequence. For example, the mutated region can be replacedby an equal number of heterologous amino acids, a smaller number ofheterologous amino acids, or a larger number of amino acids (e.g., 1-50,1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3,1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9,2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-20, 11-25,11-30, 11-35, 11-40, 11-50, 11-60, 11-70, 11-80, 11-90, 11-100, 11-150,15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90,15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80,20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100,or 30-150 amino acids). Other mutated LLO proteins have one or morepoint mutations (e.g., a point mutation of 1 residue, 2 residues, 3residues, or more). The mutated residues can be contiguous or notcontiguous.

In one example embodiment, an LLO peptide may have a deletion in thesignal sequence and a mutation or substitution in the CBD.

Some LLO peptides are N-terminal LLO fragments (i.e., LLO proteins witha C-terminal deletion). Some LLO peptides are at least 494, 489, 492,493, 500, 505, 510, 515, 520, or 525 amino acids in length or 492-528amino acids in length. For example, the LLO fragment can consist ofabout the first 440 or 441 amino acids of an LLO protein (e.g., thefirst 441 amino acids of SEQ ID NO: 55 or 56, or a correspondingfragment of another LLO protein when optimally aligned with SEQ ID NO:55 or 56). Other N-terminal LLO fragments can consist of the first 420amino acids of an LLO protein (e.g., the first 420 amino acids of SEQ IDNO: 55 or 56, or a corresponding fragment of another LLO protein whenoptimally aligned with SEQ ID NO: 55 or 56). Other N-terminal fragmentscan consist of about amino acids 20-442 of an LLO protein (e.g., aminoacids 20-442 of SEQ ID NO: 55 or 56, or a corresponding fragment ofanother LLO protein when optimally aligned with SEQ ID NO: 55 or 56).Other N-terminal LLO fragments comprise any ΔLLO without the activationdomain comprising cysteine 484, and in particular without cysteine 484.For example, the N-terminal LLO fragment can correspond to the first425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100,75, 50, or 25 amino acids of an LLO protein (e.g., the first 425, 400,375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or25 amino acids of SEQ ID NO: 55 or 56, or a corresponding fragment ofanother LLO protein when optimally aligned with SEQ ID NO: 55 or 56). Insome embodiments, the fragment comprises one or more PEST-likesequences. LLO fragments and truncated LLO proteins can contain residuesof a homologous LLO protein that correspond to any one of the abovespecific amino acid ranges. The residue numbers need not correspondexactly with the residue numbers enumerated above (e.g., if thehomologous LLO protein has an insertion or deletion relative to aspecific LLO protein disclosed herein). Examples of N-terminal LLOfragments include SEQ ID NOS: 57, 58, and 59. LLO proteins that can beused comprise, consist essentially of, or consist of the sequence setforth in SEQ ID NO: 57, 58, or 59 or homologues, variants, isoforms,analogs, fragments, fragments of homologues, fragments of variants,fragments of analogs, and fragments of isoforms of SEQ ID NO: 57, 58, or59. In some compositions and methods, the N-terminal LLO fragment setforth in SEQ ID NO: 59 is used. An example of a nucleic acid encodingthe N-terminal LLO fragment set forth in SEQ ID NO: 59 is SEQ ID NO: 60.

(2) ActA

Another example of a PEST-containing peptide that can be utilized in thecompositions and methods disclosed herein is an ActA peptide. ActA is asurface-associated protein and acts as a scaffold in infected host cellsto facilitate the polymerization, assembly, and activation of host actinpolymers in order to propel a Listeria monocytogenes through thecytoplasm. Shortly after entry into the mammalian cell cytosol, L.monocytogenes induces the polymerization of host actin filaments anduses the force generated by actin polymerization to move, firstintracellularly and then from cell to cell. ActA is responsible formediating actin nucleation and actin-based motility. The ActA proteinprovides multiple binding sites for host cytoskeletal components,thereby acting as a scaffold to assemble the cellular actinpolymerization machinery. The N-terminus of ActA binds to monomericactin and acts as a constitutively active nucleation promoting factor bystimulating the intrinsic actin nucleation activity. The actA and hlygenes are both members of the 10-kb gene cluster regulated by thetranscriptional activator PrfA, and actA is upregulated approximately226-fold in the mammalian cytosol. Any sequence that encodes an ActAprotein or a homologue, variant, isoform, analog, fragment of ahomologue, fragment of a variant, or fragment of an analog of an ActAprotein can be used. A homologous ActA protein can have a sequenceidentity with a reference ActA protein, for example, of greater than70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%,96%, 97%, 98%, or 99%.

One example of an ActA protein comprises, consists essentially of, orconsists of the sequence set forth in SEQ ID NO: 61. Another example ofan ActA protein comprises, consists essentially of, or consists of thesequence set forth in SEQ ID NO: 62. The first 29 amino acid of theproprotein corresponding to either of these sequences are the signalsequence and are cleaved from ActA protein when it is secreted by thebacterium. An ActA peptide can comprise the signal sequence (e.g., aminoacids 1-29 of SEQ ID NO: 61 or 62), or can comprise a peptide that doesnot include the signal sequence. Other examples of ActA proteinscomprise, consist essentially of, or consist of homologues, variants,isoforms, analogs, fragments, fragments of homologues, fragments ofisoforms, or fragments of analogs of SEQ ID NO: 61 or 62.

Another example of an ActA protein is an ActA protein from the Listeriamonocytogenes 10403S strain (GenBank Accession No.: DQ054585) the NICPBP54002 strain (GenBank Accession No.: EU394959), the S3 strain (GenBankAccession No.: EU394960), NCTC 5348 strain (GenBank Accession No.:EU394961), NICPBP 54006 strain (GenBank Accession No.: EU394962), M7strain (GenBank Accession No.: EU394963), S19 strain (GenBank AccessionNo.: EU394964), or any other strain of Listeria monocytogenes. LLOproteins that can be used can comprise, consist essentially of, orconsist of any of the above LLO proteins or homologues, variants,isoforms, analogs, fragments, fragments of homologues, fragments ofvariants, fragments of analogs, and fragments of isoforms of the aboveLLO proteins.

ActA peptides can be full-length ActA proteins or truncated ActAproteins or ActA fragments (e.g., N-terminal ActA fragments in which aC-terminal portion is removed). In some embodiments, truncated ActAproteins comprise at least one PEST sequence (e.g., more than one PESTsequence). In addition, truncated ActA proteins can optionally comprisean ActA signal peptide. Examples of PEST-like sequences contained intruncated ActA proteins include SEQ ID NOS: 45-48. Some such truncatedActA proteins comprise at least two of the PEST-like sequences set forthin SEQ ID NOS: 45-48 or homologs thereof, at least three of thePEST-like sequences set forth in SEQ ID NOS: 45-48 or homologs thereof,or all four of the PEST-like sequences set forth in SEQ ID NOS: 45-48 orhomologs thereof. Examples of truncated ActA proteins include thosecomprising, consisting essentially of, or consisting of about residues30-122, about residues 30-229, about residues 30-332, about residues30-200, or about residues 30-399 of a full length ActA protein sequence(e.g., SEQ ID NO: 62). Other examples of truncated ActA proteins includethose comprising, consisting essentially of, or consisting of about thefirst 50, 100, 150, 200, 233, 250, 300, 390, 400, or 418 residues of afull length ActA protein sequence (e.g., SEQ ID NO: 62). Other examplesof truncated ActA proteins include those comprising, consistingessentially of, or consisting of about residues 200-300 or residues300-400 of a full length ActA protein sequence (e.g., SEQ ID NO: 62).For example, the truncated ActA consists of the first 390 amino acids ofthe wild type ActA protein as described in U.S. Pat. No. 7,655,238,herein incorporated by reference in its entirety for all purposes. Asanother example, the truncated ActA can be an ActA-N100 or a modifiedversion thereof (referred to as ActA-N100*) in which a PEST motif hasbeen deleted and containing the nonconservative QDNKR (SEQ ID NO: 73)substitution as described in US 2014/0186387, herein incorporated byreferences in its entirety for all purposes. Alternatively, truncatedActA proteins can contain residues of a homologous ActA protein thatcorresponds to one of the above amino acid ranges or the amino acidranges of any of the ActA peptides disclosed herein. The residue numbersneed not correspond exactly with the residue numbers enumerated herein(e.g., if the homologous ActA protein has an insertion or deletion,relative to an ActA protein utilized herein, then the residue numberscan be adjusted accordingly).

Examples of truncated ActA proteins include, for example, proteinscomprising, consisting essentially of, or consisting of the sequence setforth in SEQ ID NO: 63, 64, 65, or 66 or homologues, variants, isoforms,analogs, fragments of variants, fragments of isoforms, or fragments ofanalogs of SEQ ID NO: 63, 64, 65, or 66. SEQ ID NO: 63 referred to asActA/PEST1 and consists of amino acids 30-122 of the full length ActAsequence set forth in SEQ ID NO: 62. SEQ ID NO: 64 is referred to asActA/PEST2 or LA229 and consists of amino acids 30-229 of the fulllength ActA sequence set forth in the full-length ActA sequence setforth in SEQ ID NO: 62. SEQ ID NO: 65 is referred to as ActA/PEST3 andconsists of amino acids 30-332 of the full-length ActA sequence setforth in SEQ ID NO: 62. SEQ ID NO: 66 is referred to as ActA/PEST4 andconsists of amino acids 30-399 of the full-length ActA sequence setforth in SEQ ID NO: 62. As a specific example, the truncated ActAprotein consisting of the sequence set forth in SEQ ID NO: 64 can beused.

Examples of truncated ActA proteins include, for example, proteinscomprising, consisting essentially of, or consisting of the sequence setforth in SEQ ID NO: 67, 69, 70, or 72 or homologues, variants, isoforms,analogs, fragments of variants, fragments of isoforms, or fragments ofanalogs of SEQ ID NO: 67, 69, 70, or 72. As a specific example, thetruncated ActA protein consisting of the sequence set forth in SEQ IDNO: 67 (encoded by the nucleic acid set forth in SEQ ID NO: 68) can beused. As another specific example, the truncated ActA protein consistingof the sequence set forth in SEQ ID NO: 70 (encoded by the nucleic acidset forth in SEQ ID NO: 71) can be used. SEQ ID NO: 71 is the first 1170nucleotides encoding ActA in the Listeria monocytogenes 10403S strain.In some cases, the ActA fragment can be fused to a heterologous signalpeptide. For example, SEQ ID NO: 72 sets forth an ActA fragment fused toan Hly signal peptide.

C. Generating Immunotherapy Constructs Encoding Recombinant FusionPolypeptides

Also provided herein are methods for generating immunotherapy constructsencoding or compositions comprising the recombinant fusion polypeptidesdisclosed herein. For example, such methods can comprise selecting anddesigning antigenic peptides to include in the immunotherapy construct(and, for example, testing the hydropathy of the each antigenic peptide,and modifying or deselecting an antigenic peptide if it scores above aselected hydropathy index threshold value), designing one or more fusionpolypeptides comprising each of the selected antigenic peptides, andgenerating a nucleic acid construct encoding the fusion polypeptide.

The antigenic peptides can be screened for hydrophobicity orhydrophilicity. Antigenic peptides can be selected, for example, if theyare hydrophilic or if they score up to or below a certain hydropathythreshold, which can be predictive of secretability in a particularbacteria of interest (e.g., Listeria monocytogenes). For example,antigenic peptides can be scored by Kyte and Doolittle hydropathy indexwith a 21 amino acid window, all scoring above cutoff (around 1.6) areexcluded as they are unlikely to be secretable by Listeriamonocytogenes. See, e.g., Kyte-Doolittle (1982) J Mol Biol157(1):105-132; herein incorporated by reference in its entirety for allpurposes. Alternatively, an antigenic peptide scoring about a selectedcutoff can be altered (e.g., changing the length of the antigenicpeptide). Other sliding window sizes that can be used include, forexample, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or more amino acids. Forexample, the sliding window size can be 9-11 amino acids, 11-13 aminoacids, 13-15 amino acids, 15-17 amino acids, 17-19 amino acids, 19-21amino acids, 21-23 amino acids, 23-25 amino acids, or 25-27 amino acids.Other cutoffs that can be used include, for example, the followingranges 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2.0-2.2 2.2-2.5, 2.5-3.0,3.0-3.5, 3.5-4.0, or 4.0-4.5, or the cutoff can be 1.4, 1.5, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.3, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5. Thecutoff can vary, for example, depending on the genus or species of thebacteria being used to deliver the fusion polypeptide.

Other suitable hydropathy plots or other appropriate scales include, forexample, those reported in Rose et al. (1993) Annu Rev Biomol Struct22:381-415; Biswas et al. (2003) Journal of Chromatography A1000:637-655; Eisenberg (1984) Ann Rev Biochem 53:595-623; Abraham andLeo (1987) Proteins: Structure, Function and Genetics 2:130-152; Sweetand Eisenberg (1983) Mol Biol 171:479-488; Bull and Breese (1974) ArchBiochem Biophys 161:665-670; Guy (1985) Biophys J 47:61-70; Miyazawa etal. (1985) Macromolecules 18:534-552; Roseman (1988) J Mol Biol200:513-522; Wolfenden et al. (1981) Biochemistry 20:849-855; Wilson(1981) Biochem J 199:31-41; Cowan and Whittaker (1990) Peptide Research3:75-80; Aboderin (1971) Int J Biochem 2:537-544; Eisenberg et al.(1984) J Mol Biol 179:125-142; Hopp and Woods (1981) Proc Natl Acad SciUSA 78:3824-3828; Manavalan and Ponnuswamy (1978) Nature 275:673-674;Black and Mould (1991) Anal Biochem 193:72-82; Fauchere and Pliska(1983) Eur J Med Chem 18:369-375; Janin (1979) Nature 277:491-492; Raoand Argos (1986) Biochim Biophys Acta 869:197-214; Tanford (1962) AmChem Soc 84:4240-4274; Welling et al. (1985) FEBS Lett 188:215-218;Parker et al. (1986) Biochemistry 25:5425-5431; and Cowan and Whittaker(1990) Peptide Research 3:75-80, each of which is herein incorporated byreference in its entirety for all purposes.

Optionally, the antigenic peptides can be scored for their ability tobind to the subject human leukocyte antigen (HLA) type (for example byusing the Immune Epitope Database (IED) available at www.iedb.org, whichincludes netMHCpan, ANN, SMMPMBEC. SMM, CombLib_Sidney2008, PickPocket,and netMHCcons) and ranked by best MHC binding score from each antigenicpeptide. Other sources include TEpredict(tepredict.sourceforge.net/help.html) or other available MHC bindingmeasurement scales. Cutoffs may be different for different expressionvectors such as Salmonella.

Optionally, the antigenic peptides can be screened for immunosuppressiveepitopes (e.g., T-reg epitopes, IL-10-inducing T helper epitopes, and soforth) to deselect antigenic peptides or to avoid immunosuppressiveinfluences.

Optionally, a predicative algorithm for immunogenicity of the epitopescan be used to screen the antigenic peptides. However, these algorithmsare at best 20% accurate in predicting which peptide will generate a Tcell response. Alternatively, no screening/predictive algorithms areused. Alternatively, the antigenic peptides can be screened forimmunogenicity. For example, this can comprise contacting one or more Tcells with an antigenic peptide, and analyzing for an immunogenic T cellresponse, wherein an immunogenic T cell response identifies the peptideas an immunogenic peptide. This can also comprise using an immunogenicassay to measure secretion of at least one of CD25, CD44, or CD69 or tomeasure secretion of a cytokine selected from the group comprisingIFN-γ, TNF-α, IL-1, and IL-2 upon contacting the one or more T cellswith the peptide, wherein increased secretion identifies the peptide ascomprising one or more T cell epitopes.

The selected antigenic peptides can be arranged into one or morecandidate orders for a potential fusion polypeptide. If there are moreusable antigenic peptides than can fit into a single plasmid, differentantigenic peptides can be assigned priority ranks as needed/desiredand/or split up into different fusion polypeptides (e.g., for inclusionin different recombinant Listeria strains). Priority rank can bedetermined by factors such as relative size, priority of transcription,and/or overall hydrophobicity of the translated polypeptide. Theantigenic peptides can be arranged so that they are joined directlytogether without linkers, or any combination of linkers between anynumber of pairs of antigenic peptides, as disclosed in more detailelsewhere herein. The number of linear antigenic peptides to be includedcan be determined based on consideration of the number of constructsneeded versus the mutational burden, the efficiency of translation andsecretion of multiple epitopes from a single plasmid, and the MOI neededfor each bacteria or Lm comprising a plasmid.

The combination of antigenic peptides or the entire fusion polypeptide(i.e., comprising the antigenic peptides and the PEST-containing peptideand any tags) also be scored for hydrophobicity. For example, theentirety of the fused antigenic peptides or the entire fusionpolypeptide can be scored for hydropathy by a Kyte and Doolittlehydropathy index with a sliding 21 amino acid window. If any regionscores above a cutoff (e.g., around 1.6), the antigenic peptides can bereordered or shuffled within the fusion polypeptide until an acceptableorder of antigenic peptides is found (i.e., one in which no regionscores above the cutoff). Alternatively, any problematic antigenicpeptides can be removed or redesigned to be of a different size.Alternatively or additionally, one or more linkers between antigenicpeptides as disclosed elsewhere herein can be added or modified tochange the hydrophobicity. As with hydropathy testing for the individualantigenic peptides, other window sizes can be used, or other cutoffs canbe used (e.g., depending on the genus or species of the bacteria beingused to deliver the fusion polypeptide). In addition, other suitablehydropathy plots or other appropriate scales could be used.

Optionally, the combination of antigenic peptides or the entire fusionpolypeptide can be further screened for immunosuppressive epitopes(e.g., T-reg epitopes, IL-10-inducing T helper epitopes, and so forth)to deselect antigenic peptides or to avoid immunosuppressive influences.

A nucleic acid encoding a candidate combination of antigenic peptides orfusion polypeptide can then be designed and optimized. For example, thesequence can be optimized for increased levels of translation, durationof expression, levels of secretion, levels of transcription, and anycombination thereof. For example, the increase can be 2-fold to1000-fold, 2-fold to 500-fold, 2-fold to 100-fold, 2-fold to 50-fold,2-fold to 20-fold, 2-fold to 10-fold, or 3-fold to 5-fold relative to acontrol, non-optimized sequence.

For example, the fusion polypeptide or nucleic acid encoding the fusionpolypeptide can be optimized for decreased levels of secondarystructures possibly formed in the oligonucleotide sequence, oralternatively optimized to prevent attachment of any enzyme that maymodify the sequence. Expression in bacterial cells can be hampered, forexample, by transcriptional silencing, low mRNA half-life, secondarystructure formation, attachment sites of oligonucleotide bindingmolecules such as repressors and inhibitors, and availability of raretRNAs pools. The source of many problems in bacterial expressions isfound within the original sequence. The optimization of RNAs may includemodification of cis acting elements, adaptation of its GC-content,modifying codon bias with respect to non-limiting tRNAs pools of thebacterial cell, and avoiding internal homologous regions. Thus,optimizing a sequence can entail, for example, adjusting regions of veryhigh (>80%) or very low (<30%) GC content. Optimizing a sequence canalso entail, for example, avoiding one or more of the followingcis-acting sequence motifs: internal TATA-boxes, chi-sites, andribosomal entry sites; AT-rich or GC-rich sequence stretches; repeatsequences and RNA secondary structures; (cryptic) splice donor andacceptor sites; branch points; or a combination thereof. Optimizingexpression can also entail adding sequence elements to flanking regionsof a gene and/or elsewhere in the plasmid.

Optimizing a sequence can also entail, for example, adapting the codonusage to the codon bias of host genes (e.g., Listeria monocytogenesgenes). For example, the codons that can be used for Listeriamonocytogenes include A=GCA, G=GGT, L=TTA, Q=CAA, V=GTT, C=TGT, H=CAT,M=ATG, R=CGT, W=TGG, D=GAT, I=ATT, N=AAC, S=TCT, Y=TAT, E=GAA, K=AAA,P=CCA, T=ACA, F=TTC, and STOP=TAA.

A nucleic acid encoding a fusion polypeptide can be generated andintroduced into a delivery vehicle such as a bacteria strain or Listeriastrain. Other delivery vehicles may be suitable for DNA immunotherapy orpeptide immunotherapy, such as a vaccinia virus or virus-like particle.Once a plasmid encoding a fusion polypeptide is generated and introducedinto a bacteria strain or Listeria strain, the bacteria or Listeriastrain can be cultured and characterized to confirm expression andsecretion of the fusion polypeptide comprising the antigenic peptides.

V. Immunogenic Compositions, Pharmaceutical Compositions, and Vaccines

Also provided are immunogenic compositions, pharmaceutical compositions,or vaccines comprising a lyophilized recombinant bacteria or Listeriastrain as disclosed herein, optionally wherein the lyophilizedrecombinant bacteria or Listeria strain is reconstituted by dissolvingin an amount of solvent. An immunogenic composition comprising aListeria strain can be inherently immunogenic by virtue of itscomprising a Listeria strain and/or the composition can also furthercomprise an adjuvant. Other immunogenic compositions comprise DNAimmunotherapy or peptide immunotherapy compositions.

The term “immunogenic composition” refers to any composition containingan antigen that elicits an immune response against the antigen in asubject upon exposure to the composition. The immune response elicitedby an immunogenic composition can be to a particular antigen or to aparticular epitope on the antigen.

An immunogenic composition can comprise a single lyophilized orreconstituted recombinant bacteria or Listeria strain as disclosedherein, or it can comprise multiple different lyophilized orreconstituted recombinant bacteria or Listeria strains as disclosedherein. A bacteria or Listeria strain comprising a first recombinantfusion polypeptide is different from a bacteria or Listeria straincomprising a second recombinant fusion polypeptide, for example, if thefirst recombinant fusion polypeptide includes one antigenic peptide thatthe second recombinant fusion polypeptide does not. The two recombinantfusion polypeptides can include some of the same antigenic peptides andstill be considered different. Such different lyophilized orreconstituted recombinant bacteria or Listeria strains can beadministered concomitantly to a subject or sequentially to a subject.Sequential administration can be particularly useful when a drugsubstance comprising a lyophilized or reconstituted recombinant Listeriastrain (or recombinant fusion polypeptide or nucleic acid) disclosedherein is in different dosage forms and/or is administered on differentdosing schedules (e.g., one composition from the mixture is administeredat least daily and another is administered less frequently, such as onceweekly, once every two weeks, or once every three weeks). The multiplelyophilized or reconstituted recombinant bacteria or Listeria strainscan each comprise a different set of antigenic peptides. Alternatively,two or more of the lyophilized or reconstituted recombinant bacteria orListeria strains can comprise the same set of antigenic peptides (e.g.,the same set of antigenic peptides in a different order).

An immunogenic composition can additionally comprise an adjuvant (e.g.,two or more adjuvants), a cytokine, a chemokine, or combination thereof.Optionally, an immunogenic composition can additionally comprisesantigen presenting cells (APCs), which can be autologous or can beallogeneic to the subject.

The term adjuvant includes compounds or mixtures that enhance the immuneresponse to an antigen. For example, an adjuvant can be a non-specificstimulator of an immune response or substances that allow generation ofa depot in a subject which when combined with an immunogenic compositiondisclosed herein provides for an even more enhanced and/or prolongedimmune response. An adjuvant can favor, for example, a predominantlyTh1-mediated immune response, a Th1-type immune response, or aTh1-mediated immune response. Likewise, an adjuvant can favor acell-mediated immune response over an antibody-mediated response.Alternatively, an adjuvant can favor an antibody-mediated response. Someadjuvants can enhance the immune response by slowly releasing theantigen, while other adjuvants can mediate their effects by any of thefollowing mechanisms: increasing cellular infiltration, inflammation,and trafficking to the injection site, particularly forantigen-presenting cells (APC); promoting the activation state of APCsby upregulating costimulatory signals or major histocompatibilitycomplex (MHC) expression; enhancing antigen presentation; or inducingcytokine release for indirect effect.

Examples of adjuvants include saponin QS21, CpG oligonucleotides,unmethylated CpG-containing oligonucleotides, MPL, TLR agonists, TLR4agonists, TLR9 agonists, Resiquimod®, imiquimod, cytokines or nucleicacids encoding the same, chemokines or nucleic acids encoding same,IL-12 or a nucleic acid encoding the same, IL-6 or a nucleic acidencoding the same, and lipopolysaccharides. Another example of asuitable adjuvant is Montanide ISA 51. Montanide ISA 51 contains anatural metabolizable oil and a refined emulsifier. Other examples of asuitable adjuvant include granulocyte/macrophage colony-stimulatingfactor (GM-CSF) or a nucleic acid encoding the same and keyhole limpethemocyanin (KLH) proteins or nucleic acids encoding the same. The GM-CSFcan be, for example, a human protein grown in a yeast (S. cerevisiae)vector. GM-CSF promotes clonal expansion and differentiation ofhematopoietic progenitor cells, antigen presenting cells (APCs),dendritic cells, and T cells.

Yet another example of a suitable adjuvant is detoxified listeriolysin O(dtLLO) protein. Detoxification can be accomplished by introducing pointmutations for three selected amino acids important for binding of LLO tocholesterol and for eventual membrane pore formation. The three targetedamino acids are present in the cholesterol binding domain of LLO(ECTGLAWEWWR; SEQ ID NO: 74) and can be modified in the sequence(EATGLAWEAAR; SEQ ID NO: 96) by point mutations introduced into the DNAsequence by PCR. One example of a dtLLO suitable for use as an adjuvantis encoded by SEQ ID NO: 95. The detoxified, nonhemolytic form of LLO(dtLLO) is an effective adjuvant in tumor immunotherapy and may activateinnate and cellular immune responses by acting as a PAMP. A dtLLOencoded by a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identicalto SEQ ID NO: 95 is also suitable for use as an adjuvant.

Yet other examples of adjuvants include growth factors or nucleic acidsencoding the same, cell populations, Freund's incomplete adjuvant,aluminum phosphate, aluminum hydroxide, BCG (bacille Calmette-Guerin),alum, interleukins or nucleic acids encoding the same, quill glycosides,monophosphoryl lipid A, liposomes, bacterial mitogens, bacterial toxins,or any other type of known adjuvant (see, e.g., Fundamental Immunology,5th ed. (August 2003): William E. Paul (Editor); Lippincott Williams &Wilkins Publishers; Chapter 43: Vaccines, GJV Nossal, which is hereinincorporated by reference in its entirety for all purposes).

An immunogenic composition can further comprise one or moreimmunomodulatory molecules. Examples include interferon gamma, acytokine, a chemokine, and a T cell stimulant.

An immunogenic composition can be in the form of a vaccine orpharmaceutical composition. The terms “vaccine” and “pharmaceuticalcomposition” are interchangeable and refer to an immunogenic compositionin a pharmaceutically acceptable carrier for in vivo administration to asubject. A vaccine may be, for example, a vaccine contained within anddelivered by a cell (e.g., a recombinant Listeria as disclosed herein).A vaccine may prevent a subject from contracting or developing a diseaseand/or a vaccine may be therapeutic to a subject having a disease.

A “pharmaceutically acceptable carrier” refers to a vehicle forcontaining an immunogenic composition that can be introduced into asubject without significant adverse effects and without havingdeleterious effects on the immunogenic composition. That is,“pharmaceutically acceptable” refers to any formulation which is safe,and provides the appropriate delivery for the desired route ofadministration of an effective amount of at least one immunogeniccomposition for use in the methods disclosed herein. Pharmaceuticallyacceptable carriers or vehicles or excipients are well-known.Descriptions of suitable pharmaceutically acceptable carriers, andfactors involved in their selection, are found in a variety of readilyavailable sources such as, for example, Remington's PharmaceuticalSciences, 18th ed., 1990, herein incorporated by reference in itsentirety for all purposes. Such carriers can be suitable for any routeof administration (e.g., parenteral, enteral (e.g., oral), or topicalapplication). Such pharmaceutical compositions can be buffered, forexample, wherein the pH is maintained at a particular desired value,ranging from pH 4.0 to pH 9.0, in accordance with the stability of theimmunogenic compositions and route of administration.

Suitable pharmaceutically acceptable carriers include, for example,sterile water, salt solutions such as saline, glucose, bufferedsolutions such as phosphate buffered solutions or bicarbonate bufferedsolutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose orstarch), magnesium stearate, talc, silicic acid, viscous paraffin, whiteparaffin, glycerol, alginates, hyaluronic acid, collagen, perfume oil,fatty acid monoglycerides and diglycerides, pentaerythritol fatty acidesters, hydroxy methylcellulose, polyvinyl pyrrolidone, and the like.Pharmaceutical compositions or vaccines may also include auxiliaryagents including, for example, diluents, stabilizers (e.g., sugars andamino acids), preservatives, wetting agents, emulsifiers, pH bufferingagents, viscosity enhancing additives, lubricants, salts for influencingosmotic pressure, buffers, vitamins, coloring, flavoring, aromaticsubstances, and the like which do not deleteriously react with theimmunogenic composition.

For liquid formulations (e.g., in embodiments wherein the lyophilizedrecombinant bacteria or Listeria strain is reconstituted by dissolvingin an amount of solvent), for example, pharmaceutically acceptablecarriers may be aqueous or non-aqueous solutions, suspensions,emulsions, or oils. Non-aqueous solvents include, for example, propyleneglycol, polyethylene glycol, and injectable organic esters such as ethyloleate. Aqueous carriers include, for example, water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Examples of oils include those of petroleum, animal, vegetable,or synthetic origin, such as peanut oil, soybean oil, mineral oil, oliveoil, sunflower oil, and fish-liver oil. Solid carriers/diluents include,for example, a gum, a starch (e.g., corn starch, pregelatinized starch),a sugar (e.g., lactose, mannitol, sucrose, or dextrose), a cellulosicmaterial (e.g., microcrystalline cellulose), an acrylate (e.g.,polymethylacrylate), calcium carbonate, magnesium oxide, talc, ormixtures thereof.

Optionally, sustained or directed release pharmaceutical compositions orvaccines can be formulated. This can be accomplished, for example,through use of liposomes or compositions wherein the active compound isprotected with differentially degradable coatings (e.g., bymicroencapsulation, multiple coatings, and so forth). Such compositionsmay be formulated for immediate or slow release. It is also possible tofreeze-dry the compositions and use the lyophilisates obtained (e.g.,for the preparation of products for injection).

An immunogenic composition, pharmaceutical composition, or vaccinedisclosed herein may also comprise one or more additional compoundseffective in preventing or treating cancer. For example, the additionalcompound may comprise a compound useful in chemotherapy, such asamsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil(5-FU), gemcitabine, gliadelimplants, hydroxycarbamide, idarubicin,ifosfamide, irinotecan, leucovorin, liposomaldoxorubicin,liposomaldaunorubicin, lomustine, melphalan, mercaptopurine, mesna,methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (Taxol),pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin,streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa,tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine,vinorelbine, or a combination thereof. The additional compound can alsocomprise other biologics, including Herceptin® (trastuzumab) against theHER2 antigen, Avastin® (bevacizumab) against VEGF, or antibodies to theEGF receptor, such as Erbitux® (cetuximab), and Vectibix® (panitumumab).The additional compound can also comprise, for example, an additionalimmunotherapy.

An additional compound can also comprise an immune checkpoint inhibitorantagonist, such as a PD-1 signaling pathway inhibitor, a CD-80/86 andCTLA-4 signaling pathway inhibitor, a T cell membrane protein 3 (TIM3)signaling pathway inhibitor, an adenosine A2a receptor (A2aR) signalingpathway inhibitor, a lymphocyte activation gene 3 (LAG3) signalingpathway inhibitor, a killer immunoglobulin receptor (KIR) signalingpathway inhibitor, a CD40 signaling pathway inhibitor, or any otherantigen-presenting cell/T cell signaling pathway inhibitor. Examples ofimmune checkpoint inhibitor antagonists include an anti-PD-L1/PD-L2antibody or fragment thereof, an anti-PD-1 antibody or fragment thereof,an anti-CTLA-4 antibody or fragment thereof, or an anti-B7-H4 antibodyor fragment thereof. An additional compound can also comprise a T cellstimulator, such as an antibody or functional fragment thereof bindingto a T-cell receptor co-stimulatory molecule, an antigen presenting cellreceptor binding co-stimulatory molecule, or a member of the TNFreceptor superfamily. The T-cell receptor co-stimulatory molecule cancomprise, for example, CD28 or ICOS. The antigen presenting cellreceptor binding co-stimulatory molecule can comprise, for example, aCD80 receptor, a CD86 receptor, or a CD46 receptor. The TNF receptorsuperfamily member can comprise, for example, glucocorticoid-induced TNFreceptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), orTNFR25. See, e.g., WO2016100929, WO2016011362, and WO2016011357, each ofwhich is incorporated by reference in its entirety for all purposes.

VI. Therapeutic Methods

The lyophilized bacteria or Listeria strains (optionally wherein thelyophilized recombinant bacteria or Listeria strain is reconstituted bydissolving in an amount of solvent), immunogenic compositions,pharmaceutical compositions, and vaccines disclosed herein can be usedin various methods. For example, they can be used in methods of inducingor enhancing an anti-disease-associated-antigen (e.g., cancer-associatedantigen or tumor-associated antigen) immune response in a subject, inmethods of inducing or enhancing an anti-disease (e.g., anti-tumor oranti-cancer) immune response in a subject, in methods of treating adisease (e.g., a tumor or cancer) in a subject, in methods of preventinga disease (e.g., tumor or cancer) in a subject, or in methods ofprotecting a subject against a disease (e.g., tumor or cancer). They canalso be used in methods of increasing the ratio of T effector cells toregulatory T cells (Tregs) in the spleen and tumor of a subject, whereinthe T effector cells are targeted to a disease-associated antigen. Theycan also be used in methods for increasing disease-associated-antigenantigen T cells in a subject, increasing survival time of a subjecthaving a disease, delaying the onset of a disease in a subject, oralleviating symptoms of a disease in a subject.

A method of inducing or enhancing an anti-disease-associated antigenimmune response in a subject can comprise, for example, administering tothe subject a lyophilized or reconstituted recombinant bacteria orListeria strain, an immunogenic composition, a pharmaceuticalcomposition, or a vaccine disclosed herein. An anti-disease-associatedantigen immune response can thereby be induced or enhanced in thesubject. For example, in the case of a lyophilized or reconstitutedrecombinant Listeria strain, the Listeria strain can express the fusionpolypeptide, thereby eliciting an immune response in the subject. Theimmune response can comprise, for example, a T-cell response, such as aCD4+FoxP3− T cell response, a CD8+ T cell response, or a CD4+FoxP3− andCD8+ T cell response. Such methods can also increase the ratio of Teffector cells to regulatory T cells (Tregs) in the spleen and tumormicroenvironments of the subject, allowing for a more profoundanti-tumor response in the subject.

A method of inducing or enhancing an anti-disease (e.g., anti-cancer oranti-tumor) immune response in a subject can comprise, for example,administering to the subject a lyophilized or reconstituted recombinantbacteria or Listeria strain, an immunogenic composition, apharmaceutical composition, or a vaccine disclosed herein. Ananti-disease immune response can thereby be induced or enhanced in thesubject. For example, in the case of a recombinant Listeria strain, theListeria strain can express the fusion polypeptide, thereby eliciting ananti-disease response in the subject.

A method of treating a disease (e.g., cancer or tumor) in a subject, cancomprise, for example, administering to the subject a lyophilized orreconstituted recombinant bacteria or Listeria strain, an immunogeniccomposition, a pharmaceutical composition, or a vaccine disclosedherein. The subject can then mount an immune response against thedisease expressing the disease-associated antigen, thereby treating thedisease in the subject.

A method of preventing a disease (e.g., tumor or cancer) in a subject orprotecting a subject against developing a disease, can comprise, forexample, administering to the subject a lyophilized or reconstitutedrecombinant bacteria or Listeria strain, an immunogenic composition, apharmaceutical composition, or a vaccine disclosed herein. The subjectcan then mount an immune response against the disease-associatedantigen, thereby preventing a disease or protecting the subject againstdeveloping a disease.

In some of the above methods, two or more lyophilized or reconstitutedrecombinant bacteria or Listeria strains, immunogenic compositions,pharmaceutical compositions, or vaccines are administered. The multiplerecombinant bacteria or Listeria strains, immunogenic compositions,pharmaceutical compositions, or vaccines can be administeredsequentially in any order or combination, or can be administeredsimultaneously in any combination. As an example, if four differentListeria strains are being administered, they can be administeredsequentially, they can be administered simultaneously, or they can beadministered in any combination (e.g., administering the first andsecond strains simultaneously and subsequently administering the thirdand fourth strains simultaneously). Optionally, in the case ofsequential administration, the compositions can be administered duringthe same immune response. In some embodiments the compositions areadministered within 0-10 or 3-7 days of each other. The multiplerecombinant bacteria or Listeria strains, immunogenic compositions,pharmaceutical compositions, or vaccines can each comprise a differentset of antigenic peptides. Alternatively, two or more can comprise thesame set of antigenic peptides (e.g., the same set of antigenic peptidesin a different order).

In some methods, the disease is a cancer or tumor. Cancer is aphysiological condition in mammals that is typically characterized byunregulated cell growth and proliferation. Cancers can be hematopoieticmalignancies or solid tumors (i.e., masses of cells that result fromexcessive cell growth or proliferation, including pre-cancerouslegions). Metastatic cancer refers to a cancer that has spread from theplace where it first started to another place in the body. Tumors formedby metastatic cancer cells are called a metastatic tumor or ametastasis, which is a term also used to refer to the process by whichcancer cells spread to other parts of the body. In general, metastaticcancer has the same name and same type of cancer cells as the original,or primary, cancer. Examples of solid tumors include melanoma,carcinoma, blastoma, and sarcoma. Hematologic malignancies include, forexample, leukemia or lymphoid malignancies, such as lymphoma. Exemplarycategories of cancers include brain, breast, gastrointestinal,genitourinary, gynecologic, head and neck, heme, skin and thoracic.Brain malignancies include, for example, glioblastoma, high-gradepontine glioma, low-grade glioma, medulloblastoma, neuroblastoma, andpilocytic astrocytoma. Gastrointestinal cancers include, for example,colorectal, gallbladder, hepatocellular, pancreas, PNET, gastric, andesophageal. Genitourinary cancers include, for example, adrenocortical,bladder, kidney chromophobe, renal (clear cell), renal (papillary),rhabdoid cancers, and prostate. Gynecologic cancers include, forexample, uterine carcinosarcoma, uterine endometrial, serous ovarian,and cervical. Head and neck cancers include, for example, thyroid,nasopharyngeal, head and neck, and adenoid cystic. Heme cancers include,for example, multiple myeloma, myelodysplasia, mantle-cell lymphoma,acute lymphoblastic leukemia (ALL), non-lymphoma, chronic lymphocyticleukemia (CLL), and acute myeloid leukemia (AML). Skin cancers includes,for example, cutaneous melanoma and squamous cell carcinoma. Thoraciccancers include, for example, squamous lung, small-cell lung, and lungadenocarcinoma.

More particular examples of such cancers include squamous cell cancer orcarcinoma (e.g., oral squamous cell carcinoma), myeloma, oral cancer,juvenile nasopharyngeal angiofibroma, neuroendocrine tumors, lungcancer, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioma, glioblastoma, glial tumors, cervical cancer, ovarian cancer,liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, breastcancer, triple-negative breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial cancer or uterine cancer or carcinoma,salivary gland carcinoma, kidney or renal cancer (e.g., renal cellcarcinoma), prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, fibrosarcoma, gallbladdercancer, osteosarcoma, mesothelioma, as well as head and neck cancer. Acancer can also be a brain cancer or another type of CNS or intracranialtumor. For example, a subject can have an astrocytic tumor (e.g.,astrocytoma, anaplastic astrocytoma, glioblastoma, pilocyticastrocytoma, subependymal giant cell astrocytoma, pleomorphicxanthoastrocytoma), oligodendroglial tumor (e.g., oligodendroglioma,anaplastic oligodendroglioma), ependymal cell tumor (e.g., ependymoma,anaplastic ependymoma, myxopapillary ependymoma, subependymoma), mixedglioma (e.g., mixed oligoastrocytoma, anaplastic oligoastrocytoma),neuroepithelial tumor of uncertain origin (e.g., polar spongioblastoma,astroblastoma, gliomatosis cerebri), tumor of the choroid plexus (e.g.,choroid plexus papilloma, choroid plexus carcinoma), neuronal or mixedneuronal-glial tumor (e.g., gangliocytoma, dyplastic gangliocytoma ofcerebellum, ganglioglioma, anaplastic ganglioglioma, desmoplasticinfantile ganglioma, central neurocytoma, dysembryoplasticneuroepthelial tumor, olfactory neuroblastoma), pineal parenchyma tumor(e.g., pineocytoma, pineoblastoma, mixed pineocytoma/pineoblastoma), ortumor with mixed neuroblastic or glioblastic elements (e.g.,medulloepithelioma, medulloblastoma, neuroblastoma, retinoblastoma,ependymoblastoma).

The term “treat” or “treating” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor lessen the symptoms of the targeted disease. Treating may include oneor more of directly affecting or curing, suppressing, inhibiting,preventing, reducing the severity of, delaying the onset of, slowing theprogression of, stabilizing the progression of, inducing remission of,preventing or delaying the metastasis of, reducing/ameliorating symptomsassociated with the disease, or a combination thereof. For example,treating may include increasing expected survival time. The effect(e.g., suppressing, inhibiting, preventing, reducing the severity of,delaying the onset of, slowing the progression of, stabilizing theprogression of, inducing remission of, preventing or delaying,reducing/ameliorating symptoms of, and so forth, can be relative to acontrol subject not receiving a treatment or receiving a placebotreatment. The term “treat” or “treating” can also refer to increasingpercent chance of survival or increasing expected time of survival for asubject with the disease (e.g., relative to a control subject notreceiving a treatment or receiving a placebo treatment). In one example,“treating” refers to delaying progression, expediting remission,inducing remission, augmenting remission, speeding recovery, increasingefficacy of alternative therapeutics, decreasing resistance toalternative therapeutics, or a combination thereof (e.g., relative to acontrol subject not receiving a treatment or receiving a placebotreatment). The terms “preventing” or “impeding” can refer, for exampleto delaying the onset of symptoms, preventing relapse of a disease,decreasing the number or frequency of relapse episodes, increasinglatency between symptomatic episodes, or a combination thereof. Theterms “suppressing” or “inhibiting” can refer, for example, to reducingthe severity of symptoms, reducing the severity of an acute episode,reducing the number of symptoms, reducing the incidence ofdisease-related symptoms, reducing the latency of symptoms, amelioratingsymptoms, reducing secondary symptoms, reducing secondary infections,prolonging patient survival, or a combination thereof.

The term “subject” refers to a mammal (e.g., a human) in need of therapyfor, or susceptible to developing, a disease. The term subject alsorefers to a mammal (e.g., a human) that receives either prophylactic ortherapeutic treatment. The subject may include dogs, cats, pigs, cows,sheep, goats, horses, rats, mice, non-human mammals, and humans. Theterm “subject” does not necessarily exclude an individual that ishealthy in all respects and does not have or show signs of the disease.

An individual is at increased risk of developing a disease if thesubject has at least one known risk-factor (e.g., genetic, biochemical,family history, and situational exposure) placing individuals with thatrisk factor at a statistically significant greater risk of developingthe disease than individuals without the risk factor.

A “symptom” or “sign” refers to objective evidence of a disease asobserved by a physician or subjective evidence of a disease, such asaltered gait, as perceived by the subject. A symptom or sign may be anymanifestation of a disease. Symptoms can be primary or secondary. Theterm “primary” refers to a symptom that is a direct result of aparticular disease or disorder (e.g., a tumor or cancer), while the term“secondary” refers to a symptom that is derived from or consequent to aprimary cause. The lyophilized or reconstituted recombinant bacteria orListeria strains, the immunogenic compositions, the pharmaceuticalcompositions, and the vaccines disclosed herein can treat primary orsecondary symptoms or secondary complications.

The lyophilized or reconstituted recombinant bacteria or Listeriastrains, immunogenic compositions, pharmaceutical compositions, orvaccines are administered in an effective regime, meaning a dosage,route of administration, and frequency of administration that delays theonset, reduces the severity, inhibits further deterioration, and/orameliorates at least one sign or symptom of the disease. Alternatively,the lyophilized or reconstituted recombinant bacteria or Listeriastrains, immunogenic compositions, pharmaceutical compositions, orvaccines are administered in an effective regime, meaning a dosage,route of administration, and frequency of administration that induces animmune response to a disease-associated antigen in the lyophilized orreconstituted recombinant bacteria or Listeria strain, the immunogeniccomposition, the pharmaceutical composition, or the vaccine, or thatinduces an immune response to the bacteria or Listeria strain itself. Ifa subject is already suffering from the disease, the regime can bereferred to as a therapeutically effective regime. If the subject is atelevated risk of developing the disease relative to the generalpopulation but is not yet experiencing symptoms, the regime can bereferred to as a prophylactically effective regime. In some instances,therapeutic or prophylactic efficacy can be observed in an individualpatient relative to historical controls or past experience in the samepatient. In other instances, therapeutic or prophylactic efficacy can bedemonstrated in a preclinical or clinical trial in a population oftreated patients relative to a control population of untreated patients.For example, a regime can be considered therapeutically orprophylactically effective if an individual treated patient achieves anoutcome more favorable than the mean outcome in a control population ofcomparable patients not treated by methods described herein, or if amore favorable outcome is demonstrated in treated patients versuscontrol patients in a controlled clinical trial (e.g., a phase II, phaseII/III or phase III trial) at the p<0.05 or 0.01 or even 0.001 level.

Exemplary dosages for a recombinant Listeria strain are, for example,1×10⁶-1×10⁷ CFU, 1×10⁷-1×10⁸ CFU, 1×10⁸-3.31×10¹⁰ CFU, 1×10⁹-3.31×10¹⁰CFU, 5-500×10⁸ CFU, 7-500×10⁸ CFU, 10-500×10⁸ CFU, 20-500×10⁸ CFU,30-500×10⁸ CFU, 50-500×10⁸ CFU, 70-500×10⁸ CFU, 100-500×10⁸ CFU,150-500×10⁸ CFU, 5-300×10⁸ CFU, 5-200×10⁸ CFU, 5-15×10⁸ CFU, 5-100×10⁸CFU, 5-70×10⁸ CFU, 5-50×10⁸ CFU, 5-30×10⁸ CFU, 5-20×10⁸ CFU, 1-30×10⁹CFU, 1-20×10⁹ CFU, 2-30×10⁹ CFU, 1-10×10⁹ CFU, 2-10×10⁹ CFU, 3-10×10⁹CFU, 2-7×10⁹ CFU, 2-5×10⁹ CFU, and 3-5×10⁹ CFU. Other exemplary dosagesfor a recombinant Listeria strain are, for example, 1×10⁷ organisms,1.5×10⁷ organisms, 2×10⁸ organisms, 3×10⁷ organisms, 4×10⁷ organisms,5×10⁷ organisms, 6×10⁷ organisms, 7×10⁷ organisms, 8×10⁷ organisms,10×10⁷ organisms, 1.5×10⁸ organisms, 2×10⁸ organisms, 2.5×10⁸ organisms,3×10⁸ organisms, 3.3×10⁸ organisms, 4×10⁸ organisms, 5×10⁸ organisms,1×10⁹ organisms, 1.5×10⁹ organisms, 2×10⁹ organisms, 3×10⁹ organisms,4×10⁹ organisms, 5×10⁹ organisms, 6×10⁹ organisms, 7×10⁹ organisms,8×10⁹ organisms, 10×10⁹ organisms, 1.5×10^(m) organisms, 2×10¹⁰organisms, 2.5×10^(m) organisms, 3×10¹⁰ organisms, 3.3×10¹⁰ organisms,4×10¹⁰ organisms, and 5×10¹⁰ organisms. The dosage can depend on thecondition of the patient and response to prior treatment, if any,whether the treatment is prophylactic or therapeutic, and other factors.

Administration can be by any suitable means. For example, administrationcan be parenteral, intravenous, oral, subcutaneous, intra-arterial,intracranial, intrathecal, intracerebroventricular, intraperitoneal,topical, intranasal, intramuscular, intra-ocular, intrarectal,conjunctival, transdermal, intradermal, vaginal, rectal, intratumoral,parcanceral, transmucosal, intravascular, intraventricular, inhalation(aerosol), nasal aspiration (spray), sublingual, aerosol, suppository,or a combination thereof. For intranasal administration or applicationby inhalation, solutions or suspensions of the recombinant fusionpolypeptides, nucleic acids encoding recombinant fusion polypeptides,recombinant bacteria or Listeria strains, immunogenic compositions,pharmaceutical compositions, or vaccines mixed and aerosolized ornebulized in the presence of the appropriate carrier are suitable. Suchan aerosol may comprise any lyophilized or reconstituted recombinantbacteria or Listeria strain, immunogenic composition, pharmaceuticalcomposition, or vaccine described herein. Administration may also be inthe form of a suppository (e.g., rectal suppository or urethralsuppository), in the form of a pellet for subcutaneous implantation(e.g., providing for controlled release over a period of time), or inthe form of a capsule. Administration may also be via injection into adisease site. Regimens of administration can be readily determined basedon factors such as exact nature and type of the disease being treated,the severity of the disease, the age and general physical condition ofthe subject, body weight of the subject, response of the individualsubject, and the like.

The frequency of administration can depend on the half-life of thelyophilized or reconstituted recombinant bacteria or Listeria strains,immunogenic compositions, pharmaceutical compositions, or vaccines inthe subject, the condition of the subject, and the route ofadministration, among other factors. The frequency can be, for example,daily, weekly, monthly, quarterly, or at irregular intervals in responseto changes in the subject's condition or progression of the tumor orcancer being treated. The course of treatment can depend on thecondition of the subject and other factors. For example, the course oftreatment can be several weeks, several months, or several years (e.g.,up to 2 years). For example, repeat administrations (doses) may beundertaken immediately following the first course of treatment or afteran interval of days, weeks or months to achieve disease regression orsuppression. Assessment may be determined by any known technique,including diagnostic methods such as imaging techniques, analysis ofserum biomarkers, biopsy, or the presence, absence, or amelioration ofdisease-associated symptoms. As a specific example, the lyophilized orreconstituted recombinant bacteria or Listeria strains, immunogeniccompositions, pharmaceutical compositions, or vaccines can beadministered every 3 weeks for up to 2 years. In one example, alyophilized or reconstituted recombinant bacteria or Listeria strain, animmunogenic composition, a pharmaceutical composition, or a vaccinedisclosed herein is administered in increasing doses in order toincrease the T-effector cell to regulatory T cell ratio and generate amore potent anti-disease immune response. Anti-disease immune responsescan be further strengthened by providing the subject with cytokinesincluding, for example, IFN-γ, TNF-α, and other cytokines known toenhance cellular immune response. See, e.g., U.S. Pat. No. 6,991,785,herein incorporated by reference in its entirety for all purposes.

Some methods may further comprise “boosting” the subject with additionallyophilized or reconstituted recombinant bacteria or Listeria strains,immunogenic compositions, pharmaceutical compositions, or vaccines oradministering the lyophilized or reconstituted recombinant bacteria orListeria strains, immunogenic compositions, pharmaceutical compositions,or vaccines multiple times. “Boosting” refers to administering anadditional dose to a subject. For example, in some methods, 2 boosts (ora total of 3 inoculations) are administered, 3 boosts are administered,4 boosts are administered, 5 boosts are administered, or 6 or moreboosts are administered. The number of dosages administered can dependon, for example, the response of the disease to the treatment.

Optionally, the lyophilized or reconstituted recombinant bacteria orListeria strain, immunogenic composition, pharmaceutical composition, orvaccine used in the booster inoculation is the same as the lyophilizedor reconstituted recombinant bacteria or Listeria strain, immunogeniccomposition, pharmaceutical composition, or vaccine used in the initial“priming” inoculation. Alternatively, the booster is different from thepriming recombinant bacteria or Listeria strain, immunogeniccomposition, pharmaceutical composition, or vaccine. Optionally, thesame dosages are used in the priming and boosting inoculations.Alternatively, a larger dosage is used in the booster, or a smallerdosage is used in the booster. The period between priming and boostinginoculations can be experimentally determined. For example, the periodbetween priming and boosting inoculations can be 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6-8 weeks, or 8-10 weeks.

Heterologous prime boost strategies have been effective for enhancingimmune responses and protection against numerous pathogens. See, e.g.,Schneider et al. (1999) Immunol. Rev. 170:29-38; Robinson (2002) Nat.Rev. Immunol. 2:239-250; Gonzalo et al. (2002) Vaccine 20:1226-1231; andTanghe (2001) Infect. Immun. 69:3041-3047, each of which is hereinincorporated by reference in its entirety for all purposes. Providingantigen in different forms in the prime and the boost injections canmaximize the immune response to the antigen. DNA vaccine primingfollowed by boosting with protein in adjuvant or by viral vectordelivery of DNA encoding antigen is one effective way of improvingantigen-specific antibody and CD4⁺ T-cell responses or CD8⁺ T-cellresponses. See, e.g., Shiver et al. (2002) Nature 415: 331-335; Gilbertet al. (2002) Vaccine 20:1039-1045; Billaut-Mulot et al. (2000) Vaccine19:95-102; and Sin et al. (1999) DNA Cell Biol. 18:771-779, each ofwhich is herein incorporated by reference in its entirety for allpurposes. As one example, adding CRL1005 poloxamer (12 kDa, 5% POE) toDNA encoding an antigen can enhance T-cell responses when subjects arevaccinated with a DNA prime followed by a boost with an adenoviralvector expressing the antigen. See, e.g., Shiver et al. (2002) Nature415:331-335, herein incorporated by reference in its entirety for allpurposes. As another example, a vector construct encoding an immunogenicportion of an antigen and a protein comprising the immunogenic portionof the antigen can be administered. See, e.g., US 2002/0165172, hereinincorporated by reference in its entirety for all purposes. Similarly,an immune response of nucleic acid vaccination can be enhanced bysimultaneous administration of (e.g., during the same immune response,in some embodiments within 0-10 or 3-7 days of each other) apolynucleotide and polypeptide of interest. See, e.g., U.S. Pat. No.6,500,432, herein incorporated by reference in its entirety for allpurposes.

The therapeutic methods disclosed herein can also comprise administeringone or more additional compounds effective in preventing or treating adisease (e.g., a tumor or cancer). For example, an additional compoundmay comprise a compound useful in chemotherapy, such as amsacrine,bleomycin, busulfan, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin,docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil(5-FU), gemcitabine, gliadelimplants, hydroxycarbamide, idarubicin,ifosfamide, irinotecan, leucovorin, liposomaldoxorubicin,liposomaldaunorubicin, lomustine, melphalan, mercaptopurine, mesna,methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (Taxol),pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin,streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa,tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine,vinorelbine, or a combination thereof. Alternatively, an additionalcompound can also comprise other biologics, including Herceptin®(trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) againstVEGF, or antibodies to the EGF receptor, such as Erbitux® (cetuximab),and Vectibix® (panitumumab). Alternatively, an additional compound cancomprise other immunotherapies. Alternatively, the additional compoundcan be an indoleamine 2,3-dioxygenase (IDO) pathway inhibitor, such as1-methyltryptophan (1MT), 1-methyltryptophan (1MT), Necrostatin-1,Pyridoxal Isonicotinoyl Hydrazone, Ebselen,5-Methylindole-3-carboxaldehyde, CAY10581, an anti-IDO antibody, or asmall molecule IDO inhibitor. IDO inhibition can enhance the efficacy ofchemotherapeutic agents. The therapeutic methods disclosed herein canalso be combined with radiation, stem cell treatment, surgery, or anyother treatment.

Such additional compounds or treatments can precede the administrationof a lyophilized or reconstituted recombinant bacteria or Listeriastrain, an immunogenic composition, a pharmaceutical composition, or avaccine disclosed herein, follow the administration of a lyophilized orreconstituted recombinant bacteria or Listeria strain, an immunogeniccomposition, a pharmaceutical composition, or a vaccine disclosedherein, or be simultaneous to the administration of a lyophilized orreconstituted recombinant bacteria or Listeria strain, an immunogeniccomposition, a pharmaceutical composition, or a vaccine disclosedherein.

Targeted immunomodulatory therapy is focused primarily on the activationof costimulatory receptors, for example by using agonist antibodies thattarget members of the tumor necrosis factor receptor superfamily,including 4-1BB, OX40, and GITR (glucocorticoid-induced TNFreceptor-related). The modulation of GITR has demonstrated potential inboth antitumor and vaccine settings. Another target for agonistantibodies are co-stimulatory signal molecules for T cell activation.Targeting costimulatory signal molecules may lead to enhanced activationof T cells and facilitation of a more potent immune response.Co-stimulation may also help prevent inhibitory influences fromcheckpoint inhibition and increase antigen-specific T cellproliferation.

Listeria-based immunotherapy acts by inducing the de novo generation oftumor antigen-specific T cells that infiltrate and destroy the tumor andby reducing the numbers and activities of immunosuppressive regulatory Tcells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the tumormicroenvironment. Antibodies (or functional fragments thereof) for Tcell co-inhibitory or co-stimulatory receptors (e.g., checkpointinhibitors CTLA-4, PD-1, TIM-3, LAG3 and co-stimulators CD137, OX40,GITR, and CD40) can have synergy with Listeria-based immunotherapy.

Thus, some methods can comprise further administering a compositioncomprising an immune checkpoint inhibitor antagonist, such as a PD-1signaling pathway inhibitor, a CD-80/86 and CTLA-4 signaling pathwayinhibitor, a T cell membrane protein 3 (TIM3) signaling pathwayinhibitor, an adenosine A2a receptor (A2aR) signaling pathway inhibitor,a lymphocyte activation gene 3 (LAG3) signaling pathway inhibitor, akiller immunoglobulin receptor (KIR) signaling pathway inhibitor, a CD40signaling pathway inhibitor, or any other antigen-presenting cell/T cellsignaling pathway inhibitor. Examples of immune checkpoint inhibitorantagonists include an anti-PD-L1/PD-L2 antibody or fragment thereof, ananti-PD-1 antibody or fragment thereof, an anti-CTLA-4 antibody orfragment thereof, or an anti-B7-H4 antibody or fragment thereof. Forexample, an anti PD-1 antibody can be administered to a subject at 5-10mg/kg every 2 weeks, 5-10 mg/kg every 3 weeks, 1-2 mg/kg every 3 weeks,1-10 mg/kg every week, 1-10 mg/kg every 2 weeks, 1-10 mg/kg every 3weeks, or 1-10 mg/kg every 4 weeks.

Likewise, some methods can further comprise administering a T cellstimulator, such as an antibody or functional fragment thereof bindingto a T-cell receptor co-stimulatory molecule, an antigen presenting cellreceptor binding co-stimulatory molecule, or a member of the TNFreceptor superfamily. The T-cell receptor co-stimulatory molecule cancomprise, for example, CD28 or ICOS. The antigen presenting cellreceptor binding co-stimulatory molecule can comprise, for example, aCD80 receptor, a CD86 receptor, or a CD46 receptor. The TNF receptorsuperfamily member can comprise, for example, glucocorticoid-induced TNFreceptor (GITR), OX40 (CD134 receptor), 4-1BB (CD137 receptor), orTNFR25.

For example, some methods can further comprise administering aneffective amount of a composition comprising an antibody or functionalfragment thereof binding to a T-cell receptor co-stimulatory molecule oran antibody or functional fragment thereof binding to an antigenpresenting cell receptor binding a co-stimulatory molecule. The antibodycan be, for example, an anti-TNF receptor antibody or antigen-bindingfragment thereof (e.g., TNF receptor superfamily memberglucocorticoid-induced TNF receptor (GITR), OX40 (CD134 receptor), 4-1BB(CD137 receptor), or TNFR25), an anti-OX40 antibody or antigen-bindingfragment thereof, or an anti-GITR antibody or antigen binding fragmentthereof. Alternatively, other agonistic molecules can be administered(e.g., GITRL, an active fragment of GITRL, a fusion protein containingGITRL, a fusion protein containing an active fragment of GITRL, anantigen presenting cell (APC)/T cell agonist, CD134 or a ligand orfragment thereof, CD137 or a ligand or fragment thereof, or an inducibleT cell costimulatory (ICOS) or a ligand or fragment thereof, or anagonistic small molecule).

In a specific example, some methods can further comprise administeringan anti-CTLA-4 antibody or a functional fragment thereof and/or ananti-CD137 antibody or functional fragment thereof. For example, theanti-CTLA-4 antibody or a functional fragment thereof or the anti-CD137antibody or functional fragment thereof can be administered about 72hours after the first dose of recombinant fusion polypeptide, nucleicacids encoding a recombinant fusion polypeptide, recombinant bacteria orListeria strain, immunogenic composition, pharmaceutical composition, orvaccine, or about 48 hours after the first dose of recombinant fusionpolypeptide, nucleic acids encoding a recombinant fusion polypeptide,recombinant bacteria or Listeria strain, immunogenic composition,pharmaceutical composition, or vaccine. The anti-CTLA-4 antibody or afunctional fragment thereof or anti-CD137 antibody or functionalfragment thereof can be administered at a dose, for example, of about0.05 mg/kg and about 5 mg/kg. A recombinant Listeria strain orimmunogenic composition comprising a recombinant Listeria strain can beadministered at a dose, for example, of about 1×10⁹ CFU. Some suchmethods can further comprise administering an effective amount of ananti-PD-1 antibody or functional fragment thereof.

Methods for assessing efficacy of cancer immunotherapies are well-knownand are described, for example, in Dzojic et al. (2006) Prostate66(8):831-838; Naruishi et al. (2006) Cancer Gene Ther. 13(7):658-663,Sehgal et al. (2006) Cancer Cell Int. 6:21), and Heinrich et al. (2007)Cancer Immunol Immunother 56(5):725-730, each of which is hereinincorporated by reference in its entirety for all purposes. As oneexample, for prostate cancer, a prostate cancer model can be to testmethods and compositions disclosed herein, such as a TRAMP-C2 mousemodel, a 178-2 BMA cell model, a PAIII adenocarcinoma cells model, aPC-3M model, or any other prostate cancer model.

Alternatively or additionally, the immunotherapy can be tested in humansubjects, and efficacy can be monitored using known. Such methods caninclude, for example, directly measuring CD4+ and CD8+ T cell responses,or measuring disease progression (e.g., by determining the number orsize of tumor metastases, or monitoring disease symptoms such as cough,chest pain, weight loss, and so forth). Methods for assessing theefficacy of a cancer immunotherapy in human subjects are well-known andare described, for example, in Uenaka et al. (2007) Cancer Immun. 7:9and Thomas-Kaskel et al. (2006) Int J Cancer 119(10):2428-2434, each ofwhich is herein incorporated by reference in its entirety for allpurposes.

VII. Kits

Also provided are kits comprising a reagent utilized in performing amethod disclosed herein or kits comprising a composition, tool, orinstrument disclosed herein.

For example, such kits can comprise a lyophilized recombinant bacteriaor Listeria strain disclosed herein, an immunogenic compositiondisclosed herein, a pharmaceutical composition disclosed herein, or avaccine disclosed herein. Such kits can also comprise a solvent ordiluent for reconstituting the lyophilized recombinant bacteria orListeria strain. In addition, such kits can additionally comprise aninstructional material which describes use of the lyophilizedrecombinant bacteria or Listeria strain, the immunogenic composition,the pharmaceutical composition, or the vaccine to perform the methodsdisclosed herein. Such kits can optionally further comprise anapplicator. Although model kits are described below, the contents ofother useful kits will be apparent in light of the present disclosure.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise, if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

Listing of Embodiments

The subject matter disclosed herein includes, but is not limited to, thefollowing embodiments.

1. A method for producing a lyophilized composition comprising aListeria strain, comprising: (a) providing a composition comprising aListeria strain in a formulation comprising a buffer and sucrose; (b)cooling the composition provided in step (a) at a holding temperaturebetween about −32° C. and about −80° C. in a freezing step; (c) exposingthe composition produced by step (b) to a vacuum at a holdingtemperature between about −10° C. and about −30° C. in a primary dryingstep; and (d) exposing the composition produced by step (c) to a vacuumat a holding temperature between about −5° C. and about 25° C. in asecondary drying step, whereby the lyophilized composition is produced.

2. The method of embodiment 1, wherein prior to step (a), a stressresponse is induced in the Listeria strain by exposing the Listeriastrain to a decreased temperature.

3. The method of embodiment 1, wherein prior to step (a), a stressresponse is not induced in the Listeria strain by exposing the Listeriastrain to a decreased temperature.

4. The method of any preceding embodiment, wherein the Listeria strainused in the composition in step (a) is a frozen Listeria strain that isthawed prior to step (a).

5. The method of embodiment 4, wherein the concentration of the frozenListeria strain being thawed is between about 1×10E9 to about 1×10E10colony forming units (CFU) per milliliter.

6. The method of embodiment 4 or 5, wherein the frozen Listeria strainis thawed at about 2° C. to about 37° C.

7. The method of embodiment 6, wherein the frozen Listeria strain isthawed at about 20° C. to about 37° C.

8. The method of embodiment 7, wherein the frozen Listeria strain isthawed at about 32° C. and about 37° C.

9. The method of embodiment 8, wherein the frozen Listeria strain isthawed at about 37° C.

10. The method of any one of embodiments 4-9, wherein the frozenListeria strain is thawed for no more than 8 hours.

11. The method of any one of embodiments 4-10, wherein the frozenListeria strain is held at about 2° C. to about 8° C. for no more than24 hours after thawing.

12. The method of any one of embodiments 1-3, wherein the Listeriastrain used in the composition in step (a) is freshly cultured prior tostep (a).

13. The method of any preceding embodiment, wherein the buffer is aphosphate buffer.

14. The method of any preceding embodiment, wherein the formulationcomprises about 1% to about 5% w/v sucrose.

15. The method of embodiment 14, wherein the formulation comprises about2% to about 3% w/v sucrose.

16. The method of embodiment 15, wherein the formulation comprises about2.5% w/v sucrose.

17. The method of any preceding embodiment, wherein the formulationcomprises about 1×10E9 to about 1×10E10 colony forming units (CFU) ofListeria per milliliter.

18. The method of any preceding embodiment, wherein the formulation doesnot comprise one or more of trehalose, monosodium glutamate (MSG), andrecombinant human serum albumin (rHSA).

19. The method of embodiment 18, wherein the formulation does notcomprise trehalose, MSG, or rHSA.

20. The method of any preceding embodiment, wherein the holdingtemperature in the freezing step (b) is between about −40° C. and about−50° C.

21. The method of embodiment 20, wherein the holding temperature in thefreezing step (b) is about −45° C.

22. The method of any preceding embodiment, wherein the freezing step(b) comprises decreasing the temperature to the holding temperature at arate of about 1° C. per minute.

23. The method of any preceding embodiment, wherein the cooling in thefreezing step (b) is for about 2 hours to about 4 hours.

24. The method any preceding embodiment, wherein cooling in the freezingstep (b) comprises holding the composition at the holding temperaturefor about 2 hours.

25. The method of any preceding embodiment, wherein the holdingtemperature in the primary drying step (c) is between about −12° C. andabout −22° C.

26. The method of embodiment 25, wherein the holding temperature in theprimary drying step (c) is between about −17° C. and about −19° C.

27. The method of embodiment 26, wherein the holding temperature in theprimary drying step (c) is about −18° C.

28. The method of any preceding embodiment, wherein the primary dryingstep (c) comprises increasing the temperature to the holding temperatureat a rate of about 1° C. per minute.

29. The method of any preceding embodiment, wherein the primary dryingstep (c) is for about 25 hours to about 35 hours.

30. The method of any preceding embodiment, wherein the end of theprimary drying step (c) is about 12 to about 16 hours after thecomposition has reached holding temperature.

31. The method of any preceding embodiment, wherein the primary dryingstep (c) is at a vacuum pressure of about 0.09 mbar.

32. The method of any preceding embodiment, wherein the holdingtemperature in the secondary drying step (d) is between about −5° C. andabout 20° C.

33. The method of embodiment 32, wherein the holding temperature in thesecondary drying step (d) is between about −5° C. and about 5° C.

34. The method of embodiment 33, wherein the holding temperature in thesecondary drying step (d) is about 0° C.

35. The method of any preceding embodiment, wherein the secondary dryingstep (d) comprises increasing the temperature to the holding temperatureat a rate of about 0.2° C. per minute.

36. The method of any preceding embodiment, wherein the secondary dryingstep (d) is for about 1 hour to about 10 hours.

37. The method any preceding embodiment, wherein the secondary dryingstep (d) comprises holding the composition at the holding temperaturefor about 2 hours to about 6 hours.

38. The method any embodiment 37, wherein the secondary drying step (d)comprises holding the composition at the holding temperature for about 5hours to about 6 hours.

39. The method of any preceding embodiment, wherein the secondary dryingstep (d) is at a vacuum pressure of about 0.09 mbar.

40. The method of any preceding embodiment, wherein the residualmoisture in the lyophilized composition is between about 1% and about5%.

41. The method of embodiment 40, wherein the residual moisture in thelyophilized composition is between about 2% and about 4%.

42. The method of any preceding embodiment, wherein the residualmoisture in the lyophilized composition is at least about 2.5%.

43. The method of embodiment 42, wherein the residual moisture in thelyophilized composition is at least about 3%.

44. The method of any preceding embodiment, wherein the lyophilizedcomposition shows at least about 60% viability after storage at betweenabout −20° C. and about 4° C. for about 12 months.

45. The method of embodiment 44, wherein the lyophilized compositionshows at least about 75% viability after storage at between about −20°C. and about 4° C. for about 12 months.

46. The method of embodiment 45, wherein the lyophilized compositionshows at least about 80% viability after storage at between about −20°C. and about 4° C. for about 12 months.

47. The method of any preceding embodiment, wherein the Listeria strainis a recombinant Listeria monocytogenes strain.

48. The method of any preceding embodiment, wherein the Listeria strainis a recombinant Listeria monocytogenes strain, and wherein the bufferis a phosphate buffer, and wherein the formulation comprises about 2% toabout 3% w/v sucrose, and wherein the formulation does not comprisetrehalose, MSG, or rHSA, and wherein the formulation comprises about1×10E9 to about 1×10E10 colony forming units (CFU) of Listeria permilliliter, and wherein the holding temperature in the freezing step (a)is between about −40° C. and about −50° C., and wherein the holdingtemperature in the primary drying step (c) is between −17° C. and −19°C., and wherein the holding temperature in the secondary drying step (d)is between −1° C. and 1° C., and wherein the residual moisture in thelyophilized composition is between about 2.5% and about 4%.

49. The method of 48, wherein the Listeria strain used in thecomposition in step (a) is a frozen Listeria strain that is thawed priorto step (a), and wherein the concentration of the frozen Listeria strainbeing thawed is between about 1×10E9 to about 1×10E10 colony formingunits (CFU) per milliliter, and wherein the frozen Listeria strain isthawed at about 37° C., and wherein the frozen Listeria strain is thawedfor no more than 8 hours, and wherein the frozen Listeria strain is heldat about 2° C. to about 8° C. for no more than 24 hours after thawing.

50. The method of any preceding embodiment, wherein the Listeria strainis a recombinant Listeria strain comprising a nucleic acid comprising afirst open reading frame encoding a fusion polypeptide, wherein thefusion polypeptide comprises a PEST-containing peptide fused to adisease-associated antigenic peptide.

51. The method of embodiment 50, wherein the recombinant Listeria strainis an attenuated Listeria monocytogenes strain comprising a deletion ofor inactivating mutation in prfA, wherein the nucleic acid is in anepisomal plasmid and comprises a second open reading frame encoding aD133V PrfA mutant protein.

52. The method of embodiment 50, wherein the recombinant Listeria strainis an attenuated Listeria monocytogenes strain comprising a deletion ofor inactivating mutation in actA, dal, and dat, wherein the nucleic acidis in an episomal plasmid and comprises a second open reading frameencoding an alanine racemase enzyme or a D-amino acid aminotransferaseenzyme, and wherein the PEST-containing peptide is an N-terminalfragment of LLO.

53. A formulation for lyophilization of a Listeria strain, comprising:(1) the Listeria strain; (2) a phosphate buffer; and (3) sucrose.

54. The formulation of embodiment 53, wherein the Listeria strain is astrain in which a stress response has been induced by exposing theListeria strain to a decreased temperature.

55. The formulation of embodiment 53 or 54, wherein the Listeria strainis from a frozen Listeria stock.

56. The formulation of embodiment 53 or 54, wherein the Listeria strainis from a freshly cultured Listeria stock.

57. The formulation of any one of embodiments 53-56, wherein theformulation comprises about 1% to about 5% w/v sucrose.

58. The formulation of embodiment 57, wherein the formulation comprisesabout 2% to about 3% w/v sucrose.

59. The formulation of embodiment 58, wherein the formulation comprisesabout 2.5% w/v sucrose.

60. The formulation of any one of embodiments 53-59, wherein theformulation does not comprise one or more of trehalose, monosodiumglutamate (MSG), and recombinant human serum albumin (rHSA).

61. The formulation of embodiment 60, wherein the formulation does notcomprise trehalose, MSG, or rHSA.

62. The formulation of any one of embodiments 53-61, wherein theListeria strain is a recombinant Listeria monocytogenes strain.

63. The formulation of any one of embodiments 53-62, wherein theListeria strain is a recombinant Listeria monocytogenes strain, andwherein the formulation comprises about 2% to about 3% w/v sucrose, andwherein the formulation does not comprise trehalose, MSG, or rHSA.

64. The formulation of any one of embodiments 53-63, wherein theListeria strain is a recombinant Listeria strain comprising a nucleicacid comprising a first open reading frame encoding a fusionpolypeptide, wherein the fusion polypeptide comprises a PEST-containingpeptide fused to a disease-associated antigenic peptide.

65. The formulation of embodiment 64, wherein the recombinant Listeriastrain is an attenuated Listeria monocytogenes strain comprising adeletion of or inactivating mutation in prfA, wherein the nucleic acidis in an episomal plasmid and comprises a second open reading frameencoding a D133V PrfA mutant protein.

66. The formulation of embodiment 64, wherein the recombinant Listeriastrain is an attenuated Listeria monocytogenes strain comprising adeletion of or inactivating mutation in actA, dal, and dat, wherein thenucleic acid is in an episomal plasmid and comprises a second openreading frame encoding an alanine racemase enzyme or a D-amino acidaminotransferase enzyme, and wherein the PEST-containing peptide is anN-terminal fragment of LLO.

67. A lyophilized composition produced by the method of any one ofembodiments 1-52.

68. A lyophilized composition comprising a Listeria strain, a phosphatebuffer, and sucrose.

69. The lyophilized composition of embodiment 68, wherein thelyophilized composition does not comprise one or more of trehalose,monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).

70. The lyophilized composition of embodiment 69, wherein thelyophilized composition does not comprise trehalose, MSG, or rHSA.

71. The lyophilized composition of any one of embodiments 67-70, whereinthe residual moisture in the lyophilized composition is between about 1%and about 5%.

72. The lyophilized composition of embodiment 71, wherein the residualmoisture in the lyophilized composition is between about 2% and about4%.

73. The lyophilized composition of any one of embodiments 67-72, whereinthe residual moisture in the lyophilized composition is at least about2.5%.

74. The lyophilized composition of embodiment 73, wherein the residualmoisture in the lyophilized composition is at least about 3%.

75. A lyophilized composition comprising a Listeria strain, wherein theresidual moisture in the lyophilized composition is at least about 2.5%.

76. The lyophilized composition of any one of embodiments 67-75, whereinthe lyophilized composition shows at least about 60% viability afterstorage at between about −20° C. and about 4° C. for about 12 months.

77. The lyophilized composition of embodiment 76, wherein thelyophilized composition shows at least about 75% viability after storageat between about −20° C. and about 4° C. for about 12 months.

78. The lyophilized composition of embodiment 77, wherein thelyophilized composition shows at least about 80% viability after storageat between about −20° C. and about 4° C. for about 12 months.

79. The lyophilized composition of any one of embodiments 67-78, whereinthe Listeria strain is a recombinant Listeria monocytogenes strain.

80. The lyophilized composition of any one of embodiments 67-79, whereinthe Listeria strain is a recombinant Listeria monocytogenes strain, andwherein the lyophilized composition does not comprise trehalose, MSG, orrHSA, and wherein the residual moisture in the lyophilized compositionis between 2.5% and 4%.

81. The lyophilized composition of any one of embodiments 67-80, whereinthe Listeria strain is a recombinant Listeria strain comprising anucleic acid comprising a first open reading frame encoding a fusionpolypeptide, wherein the fusion polypeptide comprises a PEST-containingpeptide fused to a disease-associated antigenic peptide.

82. The lyophilized composition of embodiment 81, wherein therecombinant Listeria strain is an attenuated Listeria monocytogenesstrain comprising a deletion of or inactivating mutation in prfA,wherein the nucleic acid is in an episomal plasmid and comprises asecond open reading frame encoding a D133V PrfA mutant protein.

83. The lyophilized composition of embodiment 81, wherein therecombinant Listeria strain is an attenuated Listeria monocytogenesstrain comprising a deletion of or inactivating mutation in actA, dal,and dat, wherein the nucleic acid is in an episomal plasmid andcomprises a second open reading frame encoding an alanine racemaseenzyme or a D-amino acid aminotransferase enzyme, and wherein thePEST-containing peptide is an N-terminal fragment of LLO.

84. A method of preparing a frozen Listeria strain for lyophilization,comprising thawing the frozen Listeria strain at a temperature betweenabout 20° C. and about 37° C.

85. The method of embodiment 84, wherein the temperature is betweenabout 32° C. and about 37° C.

86. The method of embodiment 85, wherein the temperature is about 37° C.

87. The method of any one of embodiments 84-86, wherein the frozenListeria strain is thawed for no more than 8 hours.

88. The method of any one of embodiments 84-87, wherein the frozenListeria strain is held at about 2° C. to about 8° C. for no more than24 hours after thawing.

89. The method of any one of embodiments 84-88, wherein the frozenListeria strain is thawed in a formulation comprising a buffer andsucrose.

90. The method of embodiment 89, wherein the formulation comprises about1% to about 5% w/v sucrose.

91. The method of embodiment 90, wherein the formulation comprises about2% to about 3% w/v sucrose.

92. The method of embodiment 91, wherein the formulation comprises about2.5% w/v sucrose.

93. The method of any one of embodiments 89-92, wherein the formulationdoes not comprise one or more of trehalose, monosodium glutamate (MSG),and recombinant human serum albumin (rHSA).

94. The method of embodiment 93, wherein the formulation does notcomprise trehalose, MSG, or rHSA.

95. The method of any one of embodiments 89-94, wherein the Listeriastrain is a recombinant Listeria monocytogenes strain.

96. The method of any one of embodiments 89-95, wherein the Listeriastrain is a recombinant Listeria monocytogenes strain, and wherein theformulation comprises about 2% to about 3% w/v sucrose, and wherein theformulation does not comprise trehalose, MSG, or rHSA.

The subject matter disclosed herein also includes, but is not limitedto, the following embodiments.

1. A method for producing a lyophilized composition comprising aListeria strain, comprising: (a) providing a composition comprising aListeria strain in a formulation comprising a buffer and sucrose; (b)cooling the composition provided in step (a) in a freezing step,optionally wherein the temperature is between about −32° C. and −80° C.;(c) exposing the composition produced by step (b) to a vacuum in aprimary drying step, optionally wherein the temperature is between about−10° C. and −30° C.; and (d) exposing the composition produced by step(c) to a vacuum in a secondary drying step, optionally wherein thetemperature is between about 5° C. and 25° C., optionally wherein thetemperature is between about 5° C. and 20° C., whereby the lyophilizedcomposition is produced.

2. The method of embodiment 1, wherein prior to step (a), a stressresponse is induced in the Listeria strain by exposing the Listeriastrain to a decreased temperature.

3. The method of embodiment 1, wherein prior to step (a), a stressresponse is not induced in the Listeria strain by exposing the Listeriastrain to a decreased temperature.

4. The method of any preceding embodiment, wherein the Listeria strainused in the composition in step (a) is a frozen Listeria strain that wasthawed prior to step (a).

5. The method of any one of embodiments 1-3, wherein the Listeria strainused in the composition in step (a) was freshly cultured prior to step(a).

6. The method of any preceding embodiment, wherein the buffer is aphosphate buffer.

7. The method of any preceding embodiment, wherein the formulationcomprises 1% to 5% w/v sucrose.

8. The method of embodiment 7, wherein the formulation comprises 2% to3% w/v sucrose.

9. The method of any preceding embodiment, wherein the formulation doesnot comprise one or more of trehalose, monosodium glutamate (MSG), andrecombinant human serum albumin (rHSA).

10. The method of embodiment 9, wherein the formulation does notcomprise trehalose, MSG, or rHSA.

11. The method of any preceding embodiment, wherein the temperature inthe freezing step (b) is between −40° C. and −50° C.

12. The method of any preceding embodiment, wherein the cooling in thefreezing step (b) is for 2-4 hours.

13. The method of any preceding embodiment, wherein the temperature inthe primary drying step (c) is between −12° C. and −22° C.

14. The method of embodiment 13, wherein the temperature in the primarydrying step (c) is between −17° C. and −19° C.

15. The method of any preceding embodiment, wherein the primary dryingthe primary drying step (c) is for 20-30 hours.

16. The method of any preceding embodiment, wherein the temperature inthe secondary drying step (d) is between 10° C. and 20° C.

17. The method of any preceding embodiment, wherein the secondary dryingstep (d) is for 1-10 hours.

18. The method of embodiment 17, wherein the secondary drying step (d)is for 1-3 hours.

19. The method of any preceding embodiment, wherein the residualmoisture in the lyophilized composition is between 1% and 5%.

20. The method of embodiment 19, wherein the residual moisture in thelyophilized composition is between 3% and 4%.

21. The method of any preceding embodiment, wherein the lyophilizedcomposition shows at least 60% viability after storage at −20° C. or 4°C. for 6 months.

22. The method of embodiment 21, wherein the lyophilized compositionshows at least 75% viability after storage at −20° C. or 4° C. for 6months.

23. The method of embodiment 22, wherein the lyophilized compositionshows at least 80% viability after storage at −20° C. or 4° C. for 6months.

24. The method of any preceding embodiment, wherein the Listeria strainis a recombinant Listeria monocytogenes strain.

25. The method of any preceding embodiment, wherein the Listeria strainis a recombinant Listeria monocytogenes strain, and wherein the bufferis a phosphate buffer, and wherein the formulation comprises 2% to 3%w/v sucrose, and wherein the formulation does not comprise trehalose,MSG, or rHSA, and wherein the temperature in the primary drying step (c)is between −17° C. and −19° C., wherein the temperature in the secondarydrying step (d) is between 10° C. and 20° C., and wherein the residualmoisture in the lyophilized composition is between 3% and 4%.

26. The method of any preceding embodiment, wherein the Listeria strainis a recombinant Listeria strain comprising a nucleic acid comprising afirst open reading frame encoding a fusion polypeptide, wherein thefusion polypeptide comprises a PEST-containing peptide fused to adisease-associated antigenic peptide.

27. The method of embodiment 26, wherein the recombinant Listeria strainis an attenuated Listeria monocytogenes strain comprising a deletion ofor inactivating mutation in prfA, wherein the nucleic acid is in anepisomal plasmid and comprises a second open reading frame encoding aD133V PrfA mutant protein.

28. The method of embodiment 26, wherein the recombinant Listeria strainis an attenuated Listeria monocytogenes strain comprising a deletion ofor inactivating mutation in actA, dal, and dat, wherein the nucleic acidis in an episomal plasmid and comprises a second open reading frameencoding an alanine racemase enzyme or a D-amino acid aminotransferaseenzyme, and wherein the PEST-containing peptide is an N-terminalfragment of LLO.

29. A formulation for lyophilization of a Listeria strain, comprising:(1) the Listeria strain; (2) a phosphate buffer; and (3) sucrose.

30. The formulation of embodiment 29, wherein the Listeria strain is astrain in which a stress response has been induced by exposing theListeria strain to a decreased temperature.

31. The formulation of embodiment 29 or 30, wherein the Listeria strainis from a frozen Listeria stock.

32. The formulation of embodiment 29 or 30, wherein the Listeria strainis a from a freshly cultured Listeria stock.

33. The formulation of any one of embodiments 29-32, wherein theformulation comprises 1% to 5% w/v sucrose.

34. The formulation of embodiment 33, wherein the formulation comprises2% to 3% w/v sucrose.

35. The formulation of any one of embodiments 29-34, wherein theformulation does not comprise one or more of trehalose, monosodiumglutamate (MSG), and recombinant human serum albumin (rHSA).

36. The formulation of embodiment 35, wherein the formulation does notcomprise trehalose, MSG, or rHSA.

37. The formulation of any one of embodiments 29-36, wherein theListeria strain is a recombinant Listeria monocytogenes strain.

38. The formulation of any one of embodiments 29-37, wherein theListeria strain is a recombinant Listeria monocytogenes strain, andwherein the formulation comprises 2% to 3% w/v sucrose, and wherein theformulation does not comprise trehalose, MSG, or rHSA.

39. The formulation of any one of embodiments 29-38, wherein theListeria strain is a recombinant Listeria strain comprising a nucleicacid comprising a first open reading frame encoding a fusionpolypeptide, wherein the fusion polypeptide comprises a PEST-containingpeptide fused to a disease-associated antigenic peptide.

40. The formulation of embodiment 39, wherein the recombinant Listeriastrain is an attenuated Listeria monocytogenes strain comprising adeletion of or inactivating mutation in prfA, wherein the nucleic acidis in an episomal plasmid and comprises a second open reading frameencoding a D133V PrfA mutant protein.

41. The formulation of embodiment 39, wherein the recombinant Listeriastrain is an attenuated Listeria monocytogenes strain comprising adeletion of or inactivating mutation in actA, dal, and dat, wherein thenucleic acid is in an episomal plasmid and comprises a second openreading frame encoding an alanine racemase enzyme or a D-amino acidaminotransferase enzyme, and wherein the PEST-containing peptide is anN-terminal fragment of LLO.

42. A lyophilized composition produced by the method of any one ofembodiments 1-28.

43. A lyophilized composition comprising a Listeria strain, a phosphatebuffer, and sucrose.

44. The lyophilized composition of embodiment 43, wherein thelyophilized composition does not comprise one or more of trehalose,monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).

45. The lyophilized composition of embodiment 44, wherein thelyophilized composition does not comprise trehalose, MSG, or rHSA.

46. The lyophilized composition of any one of embodiments 42-45, whereinthe residual moisture in the lyophilized composition is between 1% and5%.

47. The lyophilized composition of embodiment 46, wherein the residualmoisture in the lyophilized composition is between 2% and 4%.

48. The lyophilized composition of embodiment 47, wherein the residualmoisture in the lyophilized composition is between 3% and 4%.

49. The lyophilized composition of any one of embodiments 42-48, whereinthe lyophilized composition shows at least 60% viability after storageat −20° C. or 4° C. for 6 months.

50. The lyophilized composition of embodiment 49, wherein thelyophilized composition shows at least 75% viability after storage at−20° C. or 4° C. for 6 months.

51. The lyophilized composition of embodiment 50, wherein thelyophilized composition shows at least 80% viability after storage at−20° C. or 4° C. for 6 months.

52. The lyophilized composition of any one of embodiments 42-51, whereinthe Listeria strain is a recombinant Listeria monocytogenes strain.

53. The lyophilized composition of any one of embodiments 42-52, whereinthe Listeria strain is a recombinant Listeria monocytogenes strain, andwherein the lyophilized composition does not comprise trehalose, MSG, orrHSA, and wherein the residual moisture in the lyophilized compositionis between 3% and 4%.

54. The formulation of any one of embodiments 42-53, wherein theListeria strain is a recombinant Listeria strain comprising a nucleicacid comprising a first open reading frame encoding a fusionpolypeptide, wherein the fusion polypeptide comprises a PEST-containingpeptide fused to a disease-associated antigenic peptide.

55. The formulation of embodiment 54, wherein the recombinant Listeriastrain is an attenuated Listeria monocytogenes strain comprising adeletion of or inactivating mutation in prfA, wherein the nucleic acidis in an episomal plasmid and comprises a second open reading frameencoding a D133V PrfA mutant protein.

56. The formulation of embodiment 54, wherein the recombinant Listeriastrain is an attenuated Listeria monocytogenes strain comprising adeletion of or inactivating mutation in actA, dal, and dat, wherein thenucleic acid is in an episomal plasmid and comprises a second openreading frame encoding an alanine racemase enzyme or a D-amino acidaminotransferase enzyme, and wherein the PEST-containing peptide is anN-terminal fragment of LLO.

BRIEF DESCRIPTION OF THE SEQUENCES

The nucleotide and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three-letter code for amino acids. The nucleotidesequences follow the standard convention of beginning at the 5′ end ofthe sequence and proceeding forward (i.e., from left to right in eachline) to the 3′ end. Only one strand of each nucleotide sequence isshown, but the complementary strand is understood to be included by anyreference to the displayed strand. The amino acid sequences follow thestandard convention of beginning at the amino terminus of the sequenceand proceeding forward (i.e., from left to right in each line) to thecarboxy terminus.

TABLE 3 Description of Sequences. SEQ ID NO Type Description 1 DNASIINFEKL Tag v1 2 DNA SIINFEKL Tag v2 3 DNA SIINFEKL Tag v3 4 DNASIINFEKL Tag v4 5 DNA SIINFEKL Tag v5 6 DNA SIINFEKL Tag v6 7 DNASIINFEKL Tag v7 8 DNA SIINFEKL Tag v8 9 DNA SIINFEKL Tag v9 10 DNASIINFEKL Tag v10 11 DNA SIINFEKL Tag v11 12 DNA SIINFEKL Tag v12 13 DNASIINFEKL Tag v13 14 DNA SIINFEKL Tag v14 15 DNA SIINFEKL Tag v15 16Protein SIINFEKL Tag 17 DNA 3xFLAG Tag v1 18 DNA 3xFLAG Tag v2 19 DNA3xFLAG Tag v3 20 DNA 3xFLAG Tag v4 21 DNA 3xFLAG Tag v5 22 DNA 3xFLAGTag v6 23 DNA 3xFLAG Tag v7 24 DNA 3xFLAG Tag v8 25 DNA 3xFLAG Tag v9 26DNA 3xFLAG Tag v10 27 DNA 3xFLAG Tag v11 28 DNA 3xFLAG Tag v12 29 DNA3xFLAG Tag v13 30 DNA 3xFLAG Tag v14 31 DNA 3xFLAG Tag v15 32 Protein3xFLAG Tag 33 Protein Peptide Linker v1 34 Protein Peptide Linker v2 35Protein Peptide Linker v3 36 Protein Peptide Linker v4 37 ProteinPeptide Linker v5 38 Protein Peptide Linker v6 39 Protein Peptide Linkerv7 40 Protein Peptide Linker v8 41 Protein Peptide Linker v9 42 ProteinPeptide Linker v10 43 Protein PEST-Like Sequence v1 44 Protein PEST-LikeSequence v2 45 Protein PEST-Like Sequence v3 46 Protein PEST-LikeSequence v4 47 Protein PEST-Like Sequence v5 48 Protein PEST-LikeSequence v6 49 Protein PEST-Like Sequence v7 50 Protein PEST-LikeSequence v8 51 Protein PEST-Like Sequence v9 52 Protein PEST-LikeSequence v10 53 Protein PEST-Like Sequence v11 54 Protein PEST-LikeSequence v12 55 Protein LLO Protein v1 56 Protein LLO Protein v2 57Protein N-Terminal Truncated LLO v1 58 Protein N-Terminal Truncated LLOv2 59 Protein N-Terminal Truncated LLO v3 60 DNA Nucleic Acid EncodingN-Terminal Truncated LLO v3 61 Protein ActA Protein v1 62 Protein ActAProtein v2 63 Protein ActA Fragment v1 64 Protein ActA Fragment v2 65Protein ActA Fragment v3 66 Protein ActA Fragment v4 67 Protein ActAFragment v5 68 DNA Nucleic Acid Encoding ActA Fragment v5 69 ProteinActA Fragment v6 70 Protein ActA Fragment v7 71 DNA Nucleic AcidEncoding ActA Fragment v7 72 Protein ActA Fragment Fused to Hly SignalPeptide 73 Protein ActA Substitution 74 Protein Cholesterol-BindingDomain of LLO 75 Protein HLA-A2 restricted Epitope from NY-ESO-1 76Protein Lm Alanine Racemase 77 Protein Lm D-Amino Acid Aminotransferase78 DNA Nucleic Acid Encoding Lm Alanine Racemase 79 DNA Nucleic AcidEncoding Lm D-Amino Acid Aminotransferase 80 Protein Wild Type PrfA 81DNA Nucleic Acid Encoding Wild Type PrfA 82 Protein D133V PrfA 83 DNANucleic Acid Encoding D133V PrfA 84 DNA 4X Glycine Linker G1 85 DNA 4XGlycine Linker G2 86 DNA 4X Glycine Linker G3 87 DNA 4X Glycine LinkerG4 88 DNA 4X Glycine Linker G5 89 DNA 4X Glycine Linker G6 90 DNA 4XGlycine Linker G7 91 DNA 4X Glycine Linker G8 92 DNA 4X Glycine LinkerG9 93 DNA 4X Glycine Linker G10 94 DNA 4X Glycine Linker G11 95 ProteinDetoxified Listeriolysin O (dtLLO) 96 Protein ModifiedCholesterol-Binding Domain of dtLLO 97 Protein LLO Signal Sequence 98Protein ActA Signal Sequence 99 Protein Variant FLAG Tag 100 Protein10-Mer Peptide

EXAMPLES Example 1. Representative Drug Substance Preparation andLyophilization Cycle

The typical current storage temperature for the ADXS-HER2 and ADXS-HPV(final liquid drug products) is −80° C., which interferes with the coldchain maintenance and poses supply chain challenges. Storage of Listeriamonocytogenes final drug product (liquid) in a frozen state isinconvenient as the cold-chain must be strictly kept at all times toassure drug product efficacy and to avoid potential patient risks.ADXS-HER2, which is an attenuated, recombinant Listeria monocytogenes(Lm) transformed with a HER2/Neu fusion protein, is an Lm Technology™immunotherapy product candidate being developed to target HER2expressing cancers. Axalimogene filolisbac (ADXS-HPV) is an LmTechnology™ immunotherapy candidate developed for the treatment ofHPV-associated cancers. It is an immunotherapy based on live attenuatedListeria monocytogenes that secretes fusion protein Lm-LLO-E7 targetingHPV-associated tumors. The storage of the final drug products in afrozen liquid state is inconvenient, as the cold chain must be kept atall times to assure drug efficacy and to avoid potential patient risks.Development of a lyophilization (lyo) cycle which favors the long termstorage of the drug products at −20° C. while maintaining the cold chainwould be beneficial. Hence, a study was performed to develop alyophilization cycle which favors the long term storage of the drugproducts.

Drug Substance Process Overview for ADXS-HPV Liquid Frozen Formulations

ADXS-HPV propagation was carried out entirely within a single use closedsystem provided by rocking wave motion bioreactor technology. Thesingle-use closed system consists of a product 20 L culture bag forfermentation, a tangential flow filtration (TFF) for concentration andbuffer exchange and a product manifold for DS container filling. Each ofthese components were sterilized by gamma irradiation. The drugsubstance manufacturing process flow diagram with in-process controls isshown in FIG. 20.

1 M sodium hydroxide (NaOH) for pH control was prepared andsterile-filtered using two 0.2 μm filters in series into a 1 L pHcontrol bag. The sterilizing filters were removed by cutting through aheat-sealed section of tubing. Fermentation media and pH controlsolution were prepared per Table 4 and sterile-filtered through two 0.2μm filters in series into a sterile 5 L media addition bag. Thesterilizing filters were removed by cutting through a heat-sealedsection of tubing.

TABLE 4 Fermentation Media Formulation. Formulation Components ComponentWeights Chloramphenicol Stock Chloramphenicol 0.68 g Solution Ethanol,anhydrous 20 mL Fermentation Media (5 L) TSB QS to 5 kg D (+) Glucose32.54 g Chloramphenicol 5 mL Stock Solution pH Control Solution 1M NaOH1 L Overnight Culture TSB 100 mL Chloramphenicol 100 μL Stock GlucoseFeed D (+) Glucose 45.04 g WFI 250 mL

Diafiltration/wash buffer was prepared per Table 5 and sterile-filteredthrough two 0.2 μm filters in series into a sterile 2×10 L bag. Thesterilizing filters were removed by cutting through a heat-sealedsection of tubing.

TABLE 5 Diafiltration Wash Buffer Formulation. Formulation ComponentsComponent Weights Formulation Buffer Potassium dihydrogen 4.0 g (20 L, 2× 10 L) phosphate (KH₂PO₄), Disodium hydrogen 22.8 g orthophosphate(Na₂HPO₄) Sodium chloride (NaCl) 160 g Potassium chloride (KCl) 4.0 gSucrose 400 g Water for Injection (WFI) QS to 20.00 kg

A 20 L culture bag was pre-connected with probes for dissolved oxygenand pH monitoring. It was then aseptically filled with 5 L of growthmedium. The media addition bag was then removed by cutting through aheat-sealed section of tubing.

The wave bag was inflated with sterile-filtered compressed O₂.Sterile-filtered compressed O₂ was continuously fed during propagationat a rate of 1 L/minute and removed through an outlet port. The rockingangle was set at 10° with a rocking rate of 18 per minute.

The pH control bag and the glucose feed bag were aseptically connectedto the culture bag. During propagation, the process was automaticallymonitored and controlled for temperature, pH, and dissolved oxygen by anintegrated controlling system.

An overnight culture was initiated from the WCB by pipetting 1 mL of WCBinto 100 mL of TSB and grown for approximately 12-16 hours until anOD600 of approximately 4. Then, 100 mL of the overnight culture was usedto inoculate the production culture by aseptically transferring to theWAVE bag.

At four hours after inoculation, 200 mL of glucose was added to theculture. Growth proceeded to an OD₆₀₀ between 7.5 and 8.5. Thiscorresponds to approximately 1×10¹⁰ CFU/mL.

When the OD₆₀₀ reached the target concentration, the culture bag wasconnected, using Readymates, to the sterile TFF manifold forconcentration and diafiltration against the formulation buffer. The TFFmodule used a 0.2 μm pore size hollow fiber filter for low shearrequirements of cell separation applications.

A peristaltic pump was used to feed the fermentation culture into theTFF system primed with formulation buffer. The bulk culture in therecirculation loop was set to a flow rate of 8 L/hr. The fermentationbroth was concentrated 5-fold to a mass of approximately 1000 g.

The diafiltration/washing of the harvest concentrate was performed with≥8 diavolumes (≥8 L). The harvest DS was sampled from the TFF assemblyusing a sampling manifold welded to the TFF. Each sample bag port washeat-sealed for removal.

The OD₆₀₀ of the sample was measured and used to calculate the amount ofdilution volume needed to reach an OD₆₀₀ of 8.0±0.5. The required amountof formulation buffer was pumped into the retentate bag to dilute theharvest to the required concentration. All volume transfers werecontrolled by weight change in the respective bags. The harvest wassampled and measured to confirm the required product concentration of1×10¹⁰ CFU/mL was achieved. DS was sampled for QC analysis using thesampling manifold.

The DS was distributed into 1 L aliquots in four 1 L product bags withthe fifth bag being filled with all the remaining DS. Each bag washeat-sealed for removal from the assembly. Each bag was individuallylabeled with the appropriate information and then stored at −80±10° C.

TABLE 6 Drug Substance Processing Parameters. Control DescriptionOperating Set-Point or Range Fermentation Media pH 6.6-7.4 Rocking Rate18 Rocks/minute Rocking Angle 10° Dissolved O₂ Set Point 35%Drug Product Process Overview for ADXS-HPV Liquid Frozen Formulations

The manufacturing process of drug product (DP) involved dilution of theBDS to a final concentration of 1×10⁹ CFU/mL and aseptic filling of theformulated axalimogene filolisbac into sterile 4 mL glass vials,stoppering with 13 mm chlorobutyl stoppers and over-sealing withaluminum flip-off seals with polypropylene discs. The process flowdiagram is shown in FIG. 21.

Frozen drug substance (DS), in 1 L bags, containing up to 5 L in 1 Laliquots, was stored at −80±10° C. until manufacture of DP. DS wasthawed at ambient temperature with a target of 3 hours to initiatemanufacture of DP.

Under Grade A conditions, up to 5 L was aseptically transferred via apump into a dedicated sterile glass carboy assembly. The pooled bulkmaterial in the carboy was stirred from 80-300 rpm during the materialtransfer and was then connected to the sterile disposable filling moduleusing a sterile tube welder. For the proposed commercial process a 1:10dilution step was performed with final formulation buffer to a targetedCFU of 1×10⁹ CFU/mL.

Depyrogenated 4 mL (DIN 2R Type I borosilicate) glass vials weresemi-automatically filled by a peristaltic pump with 1.2 mL of DS usinga sterile, single use filling line and filling needle. Filled vials wereimmediately stoppered with sterilized chlorobutyl stoppers. Duringfilling, the fill volume was controlled by weight checks on 1 vial inevery 300±50 filled vials.

The finished vials were over sealed with aluminum crimp flip-off sealswith polypropylene discs. The vials were externally wiped with a 0.35%acetic acid solution and were transferred to a Grade D room for 100%visual inspection.

Vials were visually inspected for container-closure defects or atypicalappearance of product. Bulk packaged vials were stored at −80±10° C.until shipping to the labeling and packaging site.

TABLE 7 Drug Product Process Parameters Set Point or Process StageIn-Process Control Description Operating Range Bulk mixing Stirringspeed sample 190-300 rpm bottle, >3 L volume Bulk mixing Stirring speedsample 140-240 rpm bottle, 2-3 L volume Bulk mixing Stirring speedsample 100-160 rpm bottle, 1-2 L volume Bulk mixing Stirring speedsample 80-120 rpm bottle, 0-1 L volume Aseptic filling Average linespeed 1300 ± 200 vials/hr Aseptic filling Adjustment filling volume 1.2mL Aseptic filling Filling weight max. 1.279 g Aseptic filling Fillingweight min. 1.181 g Aseptic filling Reverse impulse setting 2-3(peristaltic dosing system) Aseptic filling Acceleration setting 200(peristaltic dosing system) Aseptic filling Timer delay setting 0.3 s(peristaltic dosing system) Over sealing Average line speed 300 ± 50vials/hr Over sealing Stopper safety setting 12.2 cm Over sealingCapping station vertical 9.6 cm height Over sealing Capping stationhorizontal 3.2 cm height Over sealing Closure station top dead 7.2 cmcenter Over sealing Closure station bottom 4.2 cm dead center

TABLE 8A Specifications for Axalimogene Filolisbac Drug Product. TestTest Type Acceptance Criteria Identification Western Blot In-housemethod Principal bands for HPV16-E7 conform to reference standardWestern Blot In-house method Principal bands for tLLO conform toreference standard Purity Micro- In-house method Single speciesbiological based on Ph. Eur. Examination 2.6.12, 2.6.13 Potency J774In-house method ≥1 × 10³ CFU/mL Infectivity (Cell based plate assay)Content Viable Cell In-house method 3 × 10⁸ CFU/mL- Count 5 × 10¹⁰CFU/mL General Appearance Based on USP <1> Free-flowing cream-coloredsuspension Osmolality Ph. Eur. 2.2.35 280 to 420 mOsm/kg pH Ph. Eur.2.2.3 6.0 to 7.9 Safety Endotoxin Ph. Eur. 2.6.14 ≤35 EU/mL

TABLE 8B Specifications for Axalimogene Filolisbac Drug Substance. TestTest Type Acceptance Criteria Identity Western blot In-house methodPrincipal bands for HPV16-E7¹ conform to reference standard Western blotIn-house method Principal bands for tLLO¹ conform to reference standardprfA Deletion¹ In-house method prfA gene is (PCR) not present on thechromosome prfA Mutation¹ In-house method Point mutation (PCR) in prfAgene is present on the plasmid Purity Microbiological In-house methodSingle species Examination² based on Ph. (Negative for the Eur. 2.6.12and presence of Bacterial Ph. Eur. 2.6.13 and Fungal contamination)Percentage of In-house method ≥60% Viable cells Viable cells(Fluorometric plate assay) Potency J774 Infectivity In-house method ≥1 x10³ CFU/mL (cell-based plate assay) Content Extractable Based on USP <1>≥1 mL Volume¹ Viable Cell Count In-house method 3 × 10⁸ CFU/mL- 5 × 10¹⁰CFU/mL Plasmid Copy In-house method 5 to 100 copies Number¹ GeneralAppearance Based on USP <1> Free-flowing cream- colored suspensionOsmolality¹ Ph. Eur. 2.2.35 280 to 420 mOsm/kg pH Ph. Eur. 2.2.3 6.0 to7.9 Safety Antibiotic In-House method Resistant to 50 μg/mL Sensitivity¹streptomycin Resistant to 34 μg/mL chloramphenicol Sensitive to 0.25μg/mL ampicillin Sensitive to 1 μg/mL tetracycline Sensitive to 1.5μg/mL ciprofloxacin Reversion of In-house method No detectable reversionprfA Mutation¹ (PCR) of point mutation in prfA gene on the plasmidEndotoxin² Ph. Eur. 2.6.14 ≤35 EU/mL Container Closure USP<1207> Noingress of dye Integrity³ Notes ¹Tested only at release ²Tested atrelease and at the end of shelf life ³Tested for stability only PCR =polymerase chain reaction, Ph. Eur. = European Pharmacopoeia, USP =United States Pharmacopeia.Lyophilization as an Alternative

Drug product can also be lyophilized for long-term storage. Vials withdrug product were loaded onto lyophilizer shelves that have been chilledto a refrigerated temperature, in some embodiments, about 4° C. Thechamber door was closed, and the vials were cooled to a temperature justabove the freezing point of the formulation by reducing the shelftemperature to around −4° C. and holding it there for about 30 minutes.The formulation was then frozen by ramping the shelf temperature at arate of approximately 0.5° C./min to a temperature between −40° C. and−50° C., or about −45° C., and maintaining that temperature for severalhours until all vials were frozen and the product temperatures wereclose to the shelf temperature. To conduct primary drying, the chamberwas evacuated, and the pressure was maintained at about 0.09 mbar withsterile nitrogen. The shelf temperature was raised at about 1° C./min toa temperature between −18° C. and −22° C., or about −18° C., andmaintained at that value until all product temperatures exceeded theshelf temperature for a minimum of about 10 hours. To conduct secondarydrying, the shelf temperature was raised at about 0.2° C./min to a finalvalue of 20° C. and kept there for at least 2 hours, while the pressurewas maintained at about 0.09 mbar. At the end of this secondary dryingtime, the shelf temperature was reduced to about 10° C., then thepressure was increased to about 500 mbar with nitrogen, and the vialswere stoppered within the lyophilizer. A representative lyophilizationcycle is shown in Table 9.

TABLE 9 Representative Lyophilization Cycle. Duration Shelf Temp. VacuumStep [hh:mm] [° C.] [mbar] Loading N/A 4 Off Freezing 00:20 −4 Off 00:30−4 Off 01:30 −45 Off 02:00 −45 Off Primary drying N/A −45 0.090 00:30−45 0.090 00:18 −18 0.090  26:00* −18 0.090 Secondary drying 03:10 200.090 02:00 20 0.090 End of cycle 00:10 10 0.090 N/A 10 0.090 StopperingN/A 10 500 *Until all product temperature probes have been above shelftemperature for at least 10 h.

Example 2. Optimization of Lyophilization Parameters for Listeriamonocytogenes

The ADXS Listeria monocytogenes (Lm) drug products are currentlyformulated in a phosphate buffered saline (KH₂PO4, Na₂HPO₄, KCl, NaCl)containing 2.0% sucrose with recommended storage conditions of −80° C.The ultra-low storage temperature poses challenges for cold chainmaintenance and supply chain. Hence, a development program was initiatedfor the development of a stable lyophilized drug product (DP). The goalof the program was to develop a lyophilization (Lyo) process whichfavored the long-term storage of the drug products at −20° C. or 2-8° C.A series of experiments were performed with different parameters todevelop and optimize the formulation, the pre-conditioning of cells,storage/handling of the drug substance and the lyophilization cycle.Through optimization of various parameters, a stable lyophilizedformulation was developed which has demonstrated long-term (18 months)stability at both 2-8° C. and −20° C.

Parameters tested in the experiments below include formulationparameters (buffer composition (the solution in which the cells arelyophilized in), excipient composition (the inactive substance used toaid in stability), and OD₆₀₀ at the time of lyophilization),

A series of experiments were performed with different test parameters todevelop and optimize the formulation, the pre-conditioning of cells, andthe lyophilization cycle. Formulation parameters tested included buffercomposition (the solution in which the cells are lyophilized in),excipient composition (the inactive substance used to aid in stability),and OD₆₀₀ at the time of lyophilization. Preconditioning of cellparameters tested included fresh/frozen (the storage condition of thedrug substance before lyophilization), induction of stress responseprior to lyophilization (shift in pH and/or temperature), and drugsubstance hold time/temperature (conditions at which drug substance isthawed and held prior to lyophilization). Lyophilization cycleparameters tested included primary drying shelf temperature (heat inputfor sublimation of the frozen water), secondary drying shelf temperature(heat input for desorption of moisture remaining after primary drying),and addition of an annealing step (heating the frozen formulation to atemperature below 0° C. to allow rearrangement of the ice pore structureand possibly improve primary drying). Outcomes measured to assess thesuccess of a lyophilization run included viable cell count (VCC) overtime and under different conditions (stability at −80° C., stability at2-8° C., stability at −20° C., and accelerated stability at 30° C.),residual moisture, and reconstitution time.

The experiments described below identified several findings thatappeared to enhance the stability of the lyophilized product: (1) higherresidual moisture improved the stability of the lyophilized product(WP7-Lyo4); (2) a higher shelf temperature during primary dryingimproved the stability of the lyophilized product (WP7-Lyo9); (3)preconditioning of the cells prior to lyophilization through heat shockimproved the stability of the lyophilized product (WP7-Lyo5); (4) higherVCC demonstrated slight improvement in stability of the lyophilized drugproduct relative to lower VCC (WP7, Cycle 3); and (5) the datademonstrate that the storage of the drug substance in a 1 L LDPE bag andthawing at 37° C. prior to lyophilization improved stability of thelyophilized drug product (WP7, Cycle 3). The data show that alyophilized drug product that is stable at both −20° C. and 2-8° C. longterm has been successfully developed. The resulting drug productdemonstrated good stability at both accelerated and intended storageconditions and low loss in potency due to lyophilization.

To identify and characterize lead formulations, Lyo1 and Lyo2experiments were performed, which led to the identification of twophosphate-based formulations with 5% sucrose and with 5% sucrose plusamino acid (AA) mix (final concentrations: 36 mM arginine, 57 mMglutamic acid, and 7 mM isoleucine). The characterization of these leadformulations showed their critical temperatures to be close together,allowing for the development of one cycle for both formulations. Theresidual moisture targeting and evaluation experiments, Lyo3 and Lyo4,showed the best results for higher moisture levels at a sucrose level of2.5% and allowed for the optimization of the sucrose level at 2.5% forfurther cycle development.

The three main areas of evaluation for the lyophilization cycledevelopment study included: (1) formulation development for thescreening of buffer and excipient; (2) cell culture development for thepre-conditioning of the cells prior to lyophilization; and (3)optimization of the lyophilization cycle for targeted residual moisture(RM). The series of experiments performed in the lyophilization (lyo)cycle development with the different parameters tested are summarized inTable 10.

TABLE 10 Summary of Experiments Performed in Lyophilization (Lyo) CycleDevelopment Primary Drying Secondary Drying Expt. Expt. Shelf Temp.Shelf Temp. Process Step No. Description Construct Buffer Excipients OD(° C.) (° C.) Formulation Lyo1 Buffer Screen HER2 Citrate Sucrose,Trehalose, 10 −12 20 Phosphate MSG, rHSA MOPS Lyo2 Phosphate Sucrose,rHSA, AA 10, 2 −12 20 mix (amino acid mix) Characterization HER2Phosphate 5% sucrose 10 −22 20 of Tc, Tg, and Tg′ 5% sucrose + AA mixLyo cycle Lyo3 Residual HER2 Phosphate 5% sucrose 10 −22 20 moisture2.5% sucrose targeting Lyo4 Evaluation of HER2 Phosphate 5% sucrose 10−22 20 residual 2.5% sucrose moisture on stability Cell culture/ Lyo5Evaluation of HER2 Phosphate 2.5% sucrose 10 −22 20 pre- stressconditioning treatments of cells pre- lyophilization on stability Lyo6Evaluation of HPV Phosphate 2.5% sucrose 10 −22 20 temp-shift pre-lyophilization on stability Lyo cycle Lyo7 Evaluation of HPV Phosphate2.5% sucrose 10 −30 20 temp-shift pre- lyophilization on stability Cellculture/ Lyo8 Hold time HPV Phosphate 2.5% sucrose 10 −22 pre- studyconditioni Lyo cycle Lyo9 Evaluation of HPV Phosphate 2.5% sucrose 10−18 20 increased primary drying shelf temperature Lyo10 Comparison HPVPhosphate 2.5% sucrose 10 −18 20 of plus/minus temperature shift Lyo11Stability study HPV Phosphate 2.5% sucrose 10 −18 20 without temperatureshift Cell-culture/ Lyo12 Stability study HPV Phosphate 2.5% sucrose 10−18 20 pre- of fresh vs. conditioning frozen of cells material usingdifferent thawing Cell-culture/ Lyo13 Stability study HPV Phosphate 2.5%sucrose 10 −18 20 pre- of Fresh vs conditioning Fresh/formulated ofcells material stored 3 days at 2-8° C. Lyo Cycle Lyo14 Evaluation ofHPV Phosphate 2.5% sucrose 1.5 −18 20 commercial presentation in 2Rvials Cell-culture/ Lyo15 Stability study HPV Phosphate 2.5% sucrose 1.8−18 20 Pre- of Fresh vs. conditioning Frozen of cellspellet/reconstituted material in 2R vials Expt. Temp/pH % ConditionsResults and Process Step No. Shift DS Hold RM Evaluated ConclusionsFormulation Lyo1 2-8° C., accelerated Two phosphate-based Lyo2 stabilityconditions formulations with 5% 25° C. for 3 days. sucrose and 5%sucrose + AA mix performed well. These 2 formulations were used forfurther cycle development. The critical temperatures were closetogether. Development of one cycle for both formulations. Lyo cycle Lyo3Lyo4 ~5%, 2-8° C., accelerated Best results were at ~3%, stabilityconditions higher moisture and 1% for 1, 2, and 3 levels for the sucrosedays at 30° C. level of 2.5%. The sucrose level was fixed at 2.5% andthe moisture level was fixed at 3.5%. Cell culture/ Lyo5 Group 1:control; 3.5% 2-8° C., accelerated pre- Group 2: temp-shift; stabilityconditions conditioning Group 3: pH-shift; for 1, 2, and 3 of cellsGroup 4: pH- and days at 30° C. temp-shift Lyo6 Only temp-shift, 3.5% nocontrol Lyo cycle Lyo7 Only temp-shift, 3.5% 2-8° C., −20° C., andSignificant losses on no control accelerated accelerated stabilitystability conditions with the decreased for 1, 2, and 3 shelftemperature. days at 30° C. Cell culture/ Lyo8 Part A: fresh 2-8° C.,−20° C., and Part A showed better pre- Part B: frozen acceleratedstability profile under conditioni hold stability conditions acceleratedfor 1, 2, and 3 conditions than Part days at 30° C. B. Lyo cycle Lyo9Only temp-shift, 3.5% 2-8° C., −20° C., and Improvement in no controlaccelerated accelerated stability stability conditions was observed withfor 1, 2, and 3 increased shelf days at 30° C. temperature. Lyo10 PartA: 3.0% 2-8° C., −20° C., and The results were without temp-shiftaccelerated comparable for with Part B: stability conditions and withoutthe with temp-shift for 1, 2, and 3 temperature shift. days at 30° C.Lyo11 Without temp-shift 2.5%-3.0%    2-8° C., −20° C., and acceleratedstability conditions for 1, 2, and 3 days at 30° C. Cell-culture/ Lyo12Without temp-shift Part A: fresh 2.5%-3.0%    2-8° C., −20° C., and pre-Part B: frozen, accelerated conditioning thawed at 2-8° C. stabilityconditions of cells Part C: frozen, for 1, 2, and 3 thawed at 37° C.days at 30° C. and incubated 4 h Cell-culture/ Lyo13 Without temp-shiftPart A: Fresh; 2.5-3.0% 2-8° C., −20° C. and pre- Part B: Stored 3Accelerated conditioning days at 2-8° C. conditions for 1, 2, of cellsand 3 days and 30° C. Lyo Cycle Lyo14 Without temp-shift 2.5-3.0% 2-8°C., −20° C. and Accelerated conditions for 1, 2, and 3 days and 30° C.Cell-culture/ Lyo15 Suspension A: 2.5-3.0% 2-8° C., −20° C. and Pre-Fresh; Accelerated conditioning Suspension B: conditions for 1, 2, ofcells Frozen, thawed and 3 days and at 37° C. and 30° C. resuspendedDescription of Experiments2.1. Screening and Characterization of a Lyophilization Formulation.

For the identification, optimization and characterization of 2-3 leadformulations with good stability that could be continued with duringfurther cycle development, two lyophilization experiments (Lyo1, Lyo2)were executed and 6-month stability data was generated. The leadformulations from this study were then characterized for their criticaltemperatures Tc (collapse temperature), Tg′ (glass transitiontemperature of the frozen formulation), and Tg (glass transitiontemperature of the lyophilized product).

The formulations used in Lyo1 were citrate-, phosphate- and MOPS-basedformulations. The formulations used in Lyo2 were only phosphate-basedformulations because phosphate-based formulation had better performancecompared to citrate- and MOPS-based buffers and required the smallestprocess change as they were closest to the current drug substanceformulation.

2.1.1. WP5-Lyo1.

Materials and Methods.

The ADXS-HER2 drug product was used for this study. OD₆₀₀=10 wasevaluated. The buffers and excipients used in the formulations were asfollows: Three different buffers were used in the formulations: citrate;phosphate; and MOPS (3-(N-morpholino)propanesulfonic acid). Thestabilizer mix components (excipients) used in the formulations includedvarious combinations of sucrose, trehalose, monosodium glutamate (MSG),and recombinant human serum albumin (rHSA).

Study Design.

Three different pH buffers combined with 6 different excipientcombinations resulted in 18 different formulations. For eachformulation, 20×6R vials were filled with 2 mL, resulting in a cakeheight of ˜6.54 mm. The 360 vials were randomly distributed on the 3shelves of the lyophilization machine to average out edge effects. Thelyophilization run was completed after ˜44 h and 30 min. Vials wereclosed with 0.2 μm filtered air at 600 m bar. The vials were transferredto 2° C.-8° C. storage and crimped. Residual moisture was measureddirectly after lyophilization and after 6 months storage. Viable cellcount (VCC) before and after lyophilization and the corresponding %survival data was analyzed. The spread plate method was used fordetermining the total viable cell count of microorganisms present per mLof cell culture. The medium used to perform viable cell count may varyand is determined by the growth requirements of the organism. Listeriamonocytogenes samples were cultured in Trypticase Soy Agar (TSA).

Results.

Phosphate- and citrate-based buffers yielded comparable recoveryresults. See FIG. 1. Residual moisture (RM) analysis revealed that theaddition of MSG increased the RM in all buffer systems by ˜1.0%-1.5%.Without MSG, the RM values ranged from ˜1.8% to ˜3.0%. There was noclear tendency for increase or decrease of % RM, indicating inter-assayvariability

Multivariate data analysis (MVDA) confirmed the decision to continueformulation development based on phosphate as there was no clearsuperiority of one buffer system, and phosphate requires only modestprocess change. See FIG. 1. The regression lines using stability at 4°C. showed the most consistency under the 5:0:0:0 stabilizer mixcombination (sucrose:trehalose:MSG:rHSA). See FIG. 1.

MVDA analysis also showed that in the accelerated stability study, thesamples stored at 30° C. for 3 days showed a comparable recovery as thesamples stored at 4° C. for 6 months. See FIGS. 2A and 2B. Thisindicated that higher storage temperature resulted in faster loss ofviability and that accelerated conditions might be predictable data forlong term storage at 2° C.-8° C. The lyo-cake appearance was goodoverall with no major defects or melt-backs. The sucrose basedformulations showed slight cake shrinkages at the edges (top-crown andbottom of the vials). The change in VCC at 3 days at 30° C. was similarto that seen at 6 months at 4° C. FIG. 22A shows VCC data (percent ofaverage pre-lyophilization VCC) before lyophilization andpost-lyophilization after storage at different temperatures fordifferent amounts of time in the Lyo1 experiment. Accelerated stabilityfor 3 days at 30° C. was similar to 6 months stability at 4° C. FIG. 22Bshows residual moisture immediately after lyophilization and after 6months at 2-8° C. in the Lyo1 experiment.

2.1.2. WP7-Lyo2.

Materials and Methods.

The ADXS-HER2 drug product was used for this study. OD₆₀₀ values (OD invial is representative of cell concentration) ranging from 2 to 20 wereevaluated, and two different final OD₆₀₀ values were evaluated: OD₆₀₀=10(same as Lyo1); and OD₆₀₀=2.0. The buffer used was phosphate-basedbuffer, and the stabilizer mix components (excipients) used includedsucrose, amino acid (AA) mix, and rHSA.

Study Design.

One pH buffer, 9 different excipient combinations, and 2 bacterialconcentrations resulted in 18 different formulations. The lyophilizationrun was performed similar to Lyo1. VCC was assessed on samples beforelyophilization, on frozen samples (−80° C.) before lyophilization, onsamples after lyophilization, and on samples after lyophilization andafter storage for 1, 2, 3, 6, 9, 12, or 18 months at 2-8° C., −20° C.,and −80° C.

Results.

In both the OD₆₀₀=2 and OD₆₀₀=10 groups, the highest recoveries werewith sucrose-only formulations: 2.5% in OD₆₀₀=10; and 5% sucrose inOD₆₀₀=2.0. Parallel to Lyo1, there was a correlation between formulationand residual moisture. Formulations containing sucrose-only or a mixtureof sucrose+AA had better residual moisture compared to rHSAformulations, which were drier. Percent RM at 6 months compared to afterlyophilization was increased for all samples. MVDA analysis showed thatrHSA had detrimental effects on stability. Highest recoveries (andlowest variability) were observed at lowest sucrose concentration (2.5%)and with higher OD₆₀₀ values (10 versus 2). The lyo-cake appearance wassimilar to Lyo1, and there was no significant difference between the twoOD₆₀₀ vials. FIG. 3 shows VCC data for different OD levels andstabilizer combinations (OD, stabilizer). OD levels of 2.0, 3.0, 10.0,12.5, 15.0, 17.5, and 20.0 were tested. Stabilizers including 2% and 5%sucrose were tested, optionally in combination with AA mix. VCC as apercentage of the count before lyophilization showed similar slopes atall OD levels and stabilizer combinations. Lyophilization samples storedat −20° C. for 1 month showed VCC results in between. FIG. 23A shows VCCdata (percent of average pre-lyophilization VCC) before lyophilizationand post-lyophilization after storage at different temperatures fordifferent amounts of time (months) in the Lyo2 experiment. FIG. 23Bshows residual moisture immediately after lyophilization and after 6months at 2-8° C. in the Lyo2 experiment.

TABLE 11 Summary of Conditions in FIG. 23B. OD₆₀₀ of Fill X-foldincrease in Buffer OD₆₀₀ in 2-fold #6R Volume CFU/vial compared Target #Vial Stock Vials (mL) Formulations to WP5 High OD₆₀₀ 1 12.5 25 40 3Phosphate + 1.88 (compared to Formulation 5% Sucrose OD₆₀₀ = 10) 2 12.525 40 3 Phosphate + 5% Sucrose + Amino Acid 3 10 20 40 3 Phosphate +1.50 (compared to 5% Sucrose OD₆₀₀ = 10) 4 10 20 40 3 Phosphate + 5%Sucrose + Amino Acid Low OD₆₀₀ 5 3 6 40 3 Phosphate + 2.25 (compared toFormulation 5% Sucrose OD₆₀₀ = 2) 6 3 6 40 3 Phosphate + 5% Sucrose +Amino Acid 7 2 4 40 3 Phosphate + 2.25 (compared to 5% Sucrose OD₆₀₀ =2) 8 3 4 40 3 Phosphate + 5% Sucrose + Amino Acid OD₆₀₀ 9 20 40 12 3Phosphate + 3.00 (compared to Maximum 5% Sucrose OD₆₀₀ = 10) Evaluation10 17.5 35 12 3 Phosphate + 3.63 (compared to 5% Sucrose OD₆₀₀ = 10) 1115 30 12 3 Phosphate + 2.25 (compared to 5% Sucrose OD₆₀₀ = 10)

Conclusions.

During this study, 18 different formulations in Lyo1 and 9 differentformulations in Lyo2 were analyzed. Two formulations from Lyo1 and Lyo2were identical, hence a total of 18+7=25 different formulations wereanalyzed (formulation-1 of Lyo1 is formulation-2 of Lyo2 andformulation-5 of Lyo1 is formulation-6 of Lyo2). Based on VCC resultsfrom Lyo1, optimization in Lyo2 was performed with phosphate-basedformulations based on superior performance of phosphate and only modestchange to current process. The lead formulation of Lyo1 was 5% sucrose,which performed better or equally well compared to the other buffers inLyo2. In Lyo2, an amino acid mixture was expected to exhibit additionalprotective effects during long-term storage. Both the lead formulationof Lyo1 (5% sucrose) and the formulation with amino acids were thencharacterized for their critical temperatures (Tc, Tg, and Tg′), whichwere found to be close together, allowing for the development of onesingle cycle for both formulations.

2.2. Residual Moisture Targeting.

To obtain information about the process of drying samples duringlyophilization cycle and establish correlation between secondary dryingand residual moisture content, the Lyo3 experiment was performed. Thisenabled targeting for specific residual moisture contents in futurelyophilization experiments.

2.2.1. WP5-Lyo3.

Materials and Methods.

The ADXS-HER2 drug product was used for this study. The formulationsused were phosphate-based with 2.5% sucrose, 5% sucrose, and 10%sucrose. The stabilizer mix included different combinations of sucrose,AA mix, and rHSA. Two different OD₆₀₀ values were evaluated: OD₆₀₀=10and OD₆₀₀=2.0.

Study Design.

Stability was tested at 4° C. for 13 days, 1 month, 3 months, and 6months. Accelerated stability was tested using samples stored at 13 daysat 4° C. before accelerating at 30° C. for 1 day, 2 days, or 3 days. VCCwas measured before lyophilization, VCC and RM were measured afterlyophilization and on stability, and RM was measured afterlyophilization.

To obtain information about the process of drying, the cycle was abortedat different time points to take samples to analyze residual moisture(RM). The first samples were taken directly at the end of primarydrying. A heating rate of 0.2° C./min was used in the ramp to secondarydrying, and further samples were taken directly after the ramp tosecondary drying. Secondary drying was performed at +20° C. for 8 h (3 hlonger than in Lyo2). Samples were taken every 2 h and analyzedimmediately. Based on the live RM data, secondary drying might beextended if the target of <1% is not reached.

Results/Conclusions.

As shown in FIG. 4, it is clear from the raw VCC levels that an increasein rHSA is associated with instability, counts are higher at OD₆₀₀=10,and the lowest variability of results is seen at the low level ofsucrose (2.5%). This is reinforced by 6-month data.

As shown in FIG. 5, the moisture results show little distinction basedon OD, lower RM with increased rHSA at sucrose levels of 10% and 5%, andlowest variability of results at the lowest level of sucrose (2.5%) andwithout rHSA.

These experiments also showed that material with RM within a desiredrange of ˜5% to <1% can be generated by taking the product out of thefreeze-dryer at distinct process steps, including the end of primarydrying, the end of the ramp to secondary drying, and different timepoints during secondary drying.

2.3 Evaluation of Optimal Residual Moisture on Stability.

To optimize the target residual moisture and recovery afterlyophilization, the Lyo4 stability study was performed.

2.3.1 WP7-Lyo4.

Data generated under WP7-Lyo3 demonstrated proof of principle thatmaterial with Residual Moisture within the targeted range of ˜5% to <1%can be generated by removing samples from the freeze-dryer at distinctprocess steps: (1) end of primary drying; (2) end of ramp to secondarydrying; and (3) at different time points during secondary drying. Basedon the initial data material for long-term stability studies at 2-8° C.and at 30° C., 65% RH for short-term accelerated stress stability, wasgenerated. The % residual moisture (RM) targets were ˜5%, ˜3% and about1%. Based on all data available that was available at the time materialwas generated for two lead formulations: (1) phosphate buffer, pH 7.2,5% sucrose; and (2) phosphate buffer, pH 7.2, 2.5% sucrose.

Materials and Methods.

The ADXS-HER2 drug product was used for this study. The formulationsused were phosphate-based with 2.5% sucrose and 5% sucrose. OD₆₀₀ valueswere OD₆₀₀=20 (2×10¹⁰ CFU/mL). Moisture levels were controlled byremoving the samples out of the freeze-dryer at distinct process steps:(1) end of primary drying (residual moisture target ˜5%); (2) end oframp to secondary drying (residual moisture target ˜3%); and (3) end ofsecondary drying (residual moisture target ˜1%).

Study Design.

The lyophilization run was performed similar to Lyo3 except initial ODvalue was 20. Bacteria were cultivated and concentrated bycentrifugation as described in Lyo2 to an OD₆₀₀˜20 (˜2×10¹⁰ CFU/mL).Moisture levels were controlled by where the samples were taken in thelyophilization cycle: (1) high moisture; after primary drying; moistures˜5.4-5.8%; (2) mid moisture; after ramp (immediately after the ramp ofthe shelf temperature from the primary drying temperature to thesecondary drying temperature); moistures ˜3.7-4.5%; and (3) lowmoisture; after secondary drying; moistures ˜1.1-1.3%. The first sampleswere taken directly at the end of primary drying (RM target is ˜5%), andthe second lot of samples were taken directly after the ramp tosecondary drying (RM target is ˜3%). Secondary drying was performed for12 h, after which the third lot of samples were removed (RM target is˜1%). After removal, all vials were crimped and stored at 2-8° C.(including vials for later stress stability study). RM and VCC beforelyophilization, after lyophilization, and at accelerated conditions for1, 2 and 3 days (30° C.) were analyzed. VCC titer was measured, as bothcount and percent of count before lyophilization.

Results.

Moistures achieved in this study bracket moistures seen before.Combining Lyo4 and Lyo2 studies, focusing only on sucrose 2.5% and 5.0%,and removing all reference to studies with rHSA, comparability ataccelerated conditions was seen. Best results were at higher moisturelevels for the sucrose level of 2.5%. As shown in FIG. 6, RM achieved inthis study “bracket” moistures previously seen. RM results are shown asboth the individual values and the average. The dotted lines outline therange (high and low) of results that had been seen in the long-termstudy on Lyo2. The comparability at accelerated conditions is shown inFIGS. 7A, 7B, 24A, and 24B. As shown in FIGS. 7A and 7B, the bestresults are at higher moisture levels for sucrose levels of 2.5%. FIG.24A shows residual moisture (RM) using 2.5% sucrose after primarydrying, after ramp, and after secondary drying after storage atdifferent temperatures for different amounts of time in the Lyo4experiment. FIG. 24B shows residual moisture (RM) using 5.0% sucroseafter primary drying, after ramp, and after secondary drying afterstorage at different temperatures for different amounts of time in theLyo4 experiment.

Conclusions.

This study confirmed that higher residual moisture (RM) resulted in abetter VCC profile under accelerated conditions at OD₆₀₀=20. The bestVCC profiles obtained on accelerated stability were at higher moisturelevels for the sucrose level of 2.5%. Hence, the sucrose level was fixedat 2.5% and the target residual moisture level for future developmentexperiments was targeted at 2.5-3.5%.

2.4. Evaluation of Stress Treatments Pre-Lyophilization.

To evaluate stress treatments pre-lyophilization and its effects onstability of the lyophilized material, Lyo5 was performed. This studysimulated stress conditions potentially experienced during thefermentation process.

Cold shock, heat shock, and osmotic shock to cells, may induceexpression of genes involved in the general stress response. The geneticresponse to these shock conditions are necessary for defending the cellagainst stress damage and death. Thus, greater cell survival duringlyophilization may be achieved by activating the stress response. Toevaluate the induction of a stress response in the Lm prior tolyophilization and its effects on stability of the lyophilized material,WP7-Lyo5 was performed. This study induced a stress response either by apH shift or cold shock prior to formulation and lyophilization.

2.4.1. WP7-Lyo5.

The study goal for WP7-Lyo5 was to evaluate if induction of a stressresponse (cold shock and pH shift) in the Lm could improve viability inthe reconstituted drug product.

Materials and Methods.

The ADXS-HER2 drug product was used for this study. The experimentcomprised of the following four arms: (1) Group-1: control culture; (2)Group-2: temperature-shift culture; (3) Group-3: pH-shift culture; and(4) Group-4: pH- and temperature-shift culture (pH-shift first followedby temperature-shift). To achieve the temperature-shift and pH-shift,immediately after harvesting the cells from the bioreactor, the cellswere either placed in an ice bath or the pH was dropped by the additionof acid. This induces a stress response in the cells that activates aset of genes that seem to better prepare the cells for lyophilization.The formulation was phosphate-based with 2.5% sucrose, the residualmoisture target was 3.5% and the OD₆₀₀=10 (˜1×10¹⁰ CFU/mL). 6R vialswere filled with approximately 2 mL of Drug Product. Acceleratedstability was evaluated at 30° C. for 1, 2, and 3 days.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, materialsfor group-1 (control) and group-2 (temperature-shift) were removed.Material for group-1 was processed further until formulated bulk (bulkdrug substance) was obtained, which was then stored at 2-8° C. untilvial filling. For group-2, the temperature-shift was performed in anice/salt/water bath, after which the material was stored at 2-8° C. for30 min. Then the material was processed further until formulated bulkwas obtained, which was stored at 2-8° C. until vial filling. In themeantime, the pH-shift was performed in the bioreactor using 2M HCl topH=5.25. Then the material for group-3 (pH-shift) and group-4(pH-/temp-shift) was removed. Material from group-3 was processedfurther until formulated bulk was obtained and was stored at 2-8° C.until vial filling. For group-4, the temperature-shift was performed asdescribed above and the material was stored at 2-8° C. for 30 min. Thenthe material was processed further until formulated bulk was obtained.Once formulated bulks for all groups were obtained, VCC was analyzed andlyophilization was initiated.

The lyophilization run was performed similar to Lyo4. For a targetresidual moisture of 3.5%, 2 h secondary drying time was used. VCC wasanalyzed before lyophilization, after lyophilization, and at acceleratedconditions for 1, 2, and 3 days (30° C.). VCC titer was measured,expressed as both count and percent of count before lyophilization.

Results.

The data at 3 and 6 months (and 9, 12, and 18 months) at both −20° C.and 2-8° C. demonstrate good comparability across all four groups. SeeFIG. 8 (VCC) and FIG. 25 (RM). FIG. 8 shows the percentage of VCC afterlyophilization for each experimental condition (pH shift, temperatureshift, pH/temperature shift and control) evaluated on stability at −20°C., 2-8° C., and accelerated stability. FIG. 25 shows residual moisturelevels at T=0 for each experimental condition (pH shift, temperatureshift, pH/temperature shift, and control) evaluated on stability at 2-8°C. The temperature shift condition showed a more stable profile on longterm stability compared to control or pH shift.

The data demonstrated a good stability profile both at −20° C. and 2-8°C. for the temperature shift sample relative to the other arms of thestudy. There does not appear to be a benefit to the pH shift or thepH+temperature shift pre-treatment. The data indicate thatpreconditioning the cells prior to lyophilization may increase thelong-term stability of the product. The data do not show any cleartrends in Residual Moisture across treatment arms or upon long-termstorage.

Conclusion.

This study showed that a more stable profile on long term stability andaccelerated stability conditions was obtained with the temperature shiftcondition compared to control or pH shift.

2.5. Evaluation of Temperature Shift Pre-Lyophilization

To evaluate temperature shift treatment pre-lyophilization and itseffects on stability of the lyophilized material, Lyo6 was performed.This study simulated stress conditions potentially experienced duringthe fermentation process and was a measure to condition cells for thefreezing process during lyophilization.

2.5.1. WP7-Lyo6.

Materials and Methods.

The ADXS-HPV drug product was used for this study. No control group wasincluded. The formulation was phosphate-based with 2.5% sucrose, theresidual moisture target was 3.5%, and the OD₆₀₀=10.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, therequired volume was harvested and the temperature shift was performed inan ice/salt/water bath, after which the material was stored at 2-8° C.for 30 min. Then the material was processed further until formulatedbulk was obtained. VCC was analyzed before and after the lyophilizationrun, which was performed similar to Lyo5, and at accelerated conditionsfor 1, 2, and 3 days (30° C.). VCC titer was measured, expressed both ascount and percent of count before lyophilization.

Results.

Accelerated results for ADXS-HPV were comparable to ADXS-HER2. One monthstability was consistent with accelerated stability results. See FIG. 9(VCC) and FIG. 26 (RM). The data demonstrate good stability undershort-term accelerated conditions. The data at −20° C. shows goodstability at 12 months while the 2-8° C. storage begins to show a lossin VCC after 12 months. The data show no clear trends in RM uponlong-term storage at either −20° C. or 2-8° C.

Conclusion.

This study confirmed that the lyophilized ADXS-HPV construct producedresults consistent with ADXS-HER2 and demonstrated the applicability ofthe lyophilization platform across constructs.

2.6. Evaluation of Reduced Primary Drying Shelf Temperature and ReducedFreezing Temperature.

To evaluate freezing temperature and primary drying shelf temperatureand their effects on stability of the lyophilized material, Lyo7 wasperformed.

2.6.1. WP7-Lyo7.

Materials and Methods.

The ADXS-HPV drug product was used for this study. No control group wasincluded. The formulation was phosphate-based with 2.5% sucrose, theresidual moisture target was 3.5%, and the OD₆₀₀=10. The stabilityconditions used were 2-8° C., −20° C., and accelerated conditions for 1,2, and 3 days at 30° C.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, therequired volume was harvested and the temperature-shift was performed inan ice/salt/water bath, after which the material was stored at 2-8° C.for 30 min. Then the material was processed further until formulatedbulk was obtained. The lyophilization run was performed with freezingtemperature decreased from −40° C. to −45° C., and primary drying shelftemperature was decreased from −22° C. to −30° C. VCC was analyzedbefore and after lyophilization and at accelerated conditions. VCC titerwas measured, expressed both as count and percent of count beforelyophilization.

Results.

Significant losses were observed on accelerated stability with thedecreased shelf temperature. See FIG. 10 (VCC) and FIG. 27 (RM).Decreasing shelf temperature during freezing did not improve stabilityof the lyophilized product.

2.7. Hold Time Study.

A hold time study was performed where the drug product (DP) was eitherlyophilized immediately after formulation or was frozen, thawed, andthen lyophilized. 2.7.1. WP7-Lyo8.

Materials and Methods.

The ADXS-HPV drug product was used for this study. The formulation wasphosphate-based with 2.5% sucrose, the residual moisture target was3.5%, and the OD₆₀₀ of the final formulated material=10. The stabilityconditions used were 2-8° C., −20° C., and accelerated conditions for 1,2, and 3 days at 30° C.

Study Design.

In some groups (Part A), samples were lyophilized immediately. In othergroups (Part B), samples were frozen at −80° C., thawed at 2-8° C.overnight, and then lyophilized. VCC before lyo, after lyo, and ataccelerated conditions for 1, 2 and 3 days (30° C.) was analyzed. VCCtiter was measured, expressed both as count and percent of count beforelyophilization.

Results.

Part A (continuous processing) demonstrated a better stability profileunder accelerated conditions compared to Part B (frozen hold). See FIG.11 (VCC) and FIG. 28 (RM).

2.8. Evaluation of Increased Primary Drying Shelf Temperature.

To evaluate an increased shelf temperature during primary drying and itseffect on stability of the lyophilized material, Lyo9 was performed.

2.8.1. WP7-Lyo9.

Based on previous observations that a lower primary drying shelftemperature reduced the stability of the resulting drug product, thegoal of WP7-Lyo9 was to evaluate the effect of a higher shelftemperature during primary drying on the stability of freshly(continuously processed) lyophilized drug substance ADXS11-001 (HPV). Ashelf-temperature of −18° C. during primary drying was evaluated.Lyophilized material was staged on stability at 2-8° C. and −20° C.Further accelerated stability for 1, 2, and 3 days at 30° C./65% RH.

Materials and Methods.

The ADXS-HPV drug product was used for this study. A shelf temperatureof −18° C. was evaluated. The temperature shift was performed similar toprior experiments. The formulation was phosphate-based with 2.5%sucrose, the residual moisture target was 3.5%, and the OD₆₀₀=10. Thestability conditions used were 2-8° C., −20° C., and acceleratedconditions for 1, 2, and 3 days at 30° C.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, therequired volume was harvested and the temperature-shift was performed inan ice/salt/water bath, after which the material was stored at 2-8° C.for 30 min. Then the material was processed further until formulatedbulk was obtained. The lyophilization run was performed with a primarydrying shelf temperature of −18° C. VCC was analyzed before and afterlyophilization and at accelerated conditions. VCC titer was measured,expressed both as count and percent of count before lyophilization.

Results.

FIG. 12 shows % VCC after lyophilization evaluated on stability at 30°C., −20° C. and 2-8° C. FIG. 29 shows residual moisture levels evaluatedon stability at 30° C., −20° C. and 2-8°. The data demonstrates thatboth −20° C. and 2-8° C. are stable up to 12 months. An improvement inaccelerated stability was observed with increased shelf temperature. SeeFIG. 12 (VCC) and FIG. 29 (RM). In general, lyophilization of proteinsat primary drying shelf temperatures high enough to cause the type ofcollapse seen in this study leads to reduced stability in thelyophilized cake. However, the trend for this whole bacteria formulationappears to be the opposite.

Conclusion.

This study showed that increased primary drying shelf temperatureresulted in improvement in accelerated and long-term stability for thelyophilized product.

2.9 Comparison of Plus/Minus Temperature Shift at Increased PrimaryDrying Temperature (−18° C.).

To compare the minus [Part-A]/plus [Part-B] temperature shift atelevated primary drying temperature of Ts=−18° C., the Lyo10 stabilitystudy was performed.

2.9.1. WP7-Lyo10.

Materials and Methods.

The ADXS-HPV drug product was used for this study. A shelf temperatureof −18° C. was evaluated for two groups of materials: (1) Part-Amaterial—processed immediately after harvest (without temperatureshift); and (2) Part-B material—temperature shift was performed.

The formulation was phosphate-based with 2.5% sucrose, the residualmoisture target was 3.0%, and the OD₆₀₀=10. The stability conditionsused were 2-8° C., −20° C. and accelerated conditions for 1, 2, and 3days at 30° C.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, therequired volume for both Part-A and Part-B was harvested. Part-A wasimmediately processed until vial filling and for Part-B,temperature-shift was performed in an ice/salt/water bath, after whichthe material was stored at 2-8° C. for 30 min. Then the Part-A andPart-B materials were processed further until formulated bulk wasobtained. The lyophilization run was performed with a primary dryingshelf temperature of −18° C. and 2 h secondary drying time to target aresidual moisture of 3.0%. VCC was analyzed before and afterlyophilization and at accelerated conditions. VCC titer was measured,expressed both as count and percent of count before lyophilization.

Results.

With the increased shelf temperature, the results were comparable withand without the temperature shift. See FIG. 13 (VCC) and FIG. 30 (RM).The results confirmed the good stability profile previously observed forthe increased shelf temperature.

2.10. Stability Study without Temperature Shift at Primary DryingTemperature of −18° C. and Bioactivity Determination.

To confirm the results of Lyo10, a Lyo11 stability study was performedwithout temperature shift at elevated primary drying temperature of −18°C. Bioactivity of the lyophilized drug product was then compared toliquid frozen drug product.

2.10.1. WP7-Lyo11.

Materials and Methods.

The ADXS-HPV drug product was used for this study. A shelf temperatureof −18° C. was evaluated without temperature shift. The formulation wasphosphate-based with 2.5% sucrose, the residual moisture target was2.5-3.0%, and the OD₆₀₀=10. The stability conditions used were 2-8° C.,−20° C. and accelerated conditions for 1, 2, and 3 days at 30° C.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, therequired volume was harvested and immediately processed until formulatedbulk was obtained. The lyophilization run was performed with a primarydrying shelf temperature of −18° C., and a residual moisture of 2.5-3.0%was targeted. VCC was analyzed before and after lyophilization and ataccelerated conditions. VCC titer was measured, expressed both as countand percent of count before lyophilization.

Results.

The data show that the temperature shift is not needed in order toachieve acceptable results when the shelf temperature is increased. SeeFIG. 14 (VCC) and FIG. 31 (RM). Lyophilized samples are stable out to 12months for both 2-8° C. and −20° C. storage.

Advs11 frozen formulation (5271-15-01), lyo11 (5277 WP7 Lyo11)lyophilized formulation, and XFL7-tLLO-negative control strain were usedto infect THP1 cells at MOI of 2 and 20. 10-mer peptide (YMLDLQPETT, SEQID NO: 100) was used as a positive control. Infected cells were theninvocated for 20-24 hours, collected, and combined with T cells specificfor the 10-mer peptide. After 18-24 hours, T cell IFNγ secretion wasdetermined. A t low MOI, lyophilized formulation induced higher IFNγproduction in the T cells. At higher MOI, lyophilized formulation showedsimilar induction of IFNγ production. The percent live for thelyophilized formulation was 95%. No loss in bioactivity was observed forthe lyophilized product and for low MOI, bioactivity was increased. SeeFIG. 39.

2.11. Stability Study of Fresh Vs. Frozen Material with DifferentThawing

To confirm the results obtained from Lyo8, Lyo10, and Lyo11, a stabilitystudy Lyo12 was performed where a comparison was made between fresh andfrozen material with the frozen material being thawed in different ways.

2.11.1. WP7-Lyo12.

Materials and Methods.

The ADXS-HPV drug product was used for this study, and no temperatureshift was performed. The formulation was phosphate-based with 2.5%sucrose, the residual moisture target was 2.5-3.0%, and the OD₆₀₀=10.The stability conditions used were 2-8° C., −20° C. and acceleratedconditions for 1, 2, and 3 days at 30° C. The groups tested were: (1)Group A: control, lyophilized directly; (2) Group B: frozen at <−70° C.,thawed at 2-8° C.; and (3) Group C: frozen at <−70° C., thawed at 37° C.in water bath, then incubated for 4 h at 37° C.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, materialwas harvested and split into 3 aliquots for Parts A, B and C. Part A wasimmediately processed until formulated bulk was obtained and VCCanalysis was performed before lyophilization. Part B and Part Cmaterials were processed, aliquoted and frozen at <−70° C. Part Bmaterial was thawed at 2-8° C. overnight and Part C material was thawedcompletely in a water bath at 37° C. and was then incubated in the waterbath at 37° C. for 4 h before lyophilization. Materials were thendiluted to OD₆₀₀=10, and processed for lyophilization. Thelyophilization run was performed for a target residual moisture of2.5-3.0%. VCC was analyzed before and after lyophilization and ataccelerated conditions. VCC titer was measured, expressed both as countand percent of count before lyophilization.

Results.

The data show that continually processed material has the betterstability profile compare to frozen and thawed material. The data alsodemonstrate the drug substance may be stored prior to lyophilization.See FIG. 15 (VCC) and FIG. 32 (RM).

2.11.2. WP7-Lyo13.

Materials and Methods.

The ADXS-HPV drug product was used for this study. The formulation wasphosphate-based with 2.5% sucrose, the residual moisture target was2.5-3.0%, and the OD₆₀₀=10. The stability conditions used were 2-8° C.,−20° C. and accelerated conditions for 1, 2, and 3 days at 30° C. Thegroups tested were: (1) Group A: fresh, lyophilized directly; (2) GroupB: stored at 2-8° C. for 3 days.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, materialwas harvested and split into 2 aliquots for Parts A and B. Part A wasimmediately processed until formulated bulk was obtained and VCCanalysis was performed before lyophilization. Part B materials wereprocessed, aliquoted and frozen at <−70° C. Part B material was storedat 2-8° C. for 3 days before lyophilization. Materials were then dilutedto OD₆₀₀=10, and processed for lyophilization. The lyophilization runwas performed for a target residual moisture of 2.5-3.0%. VCC wasanalyzed before and after lyophilization and at accelerated conditions.VCC titer was measured, expressed both as count and percent of countbefore lyophilization.

Results.

The data show good results for both continually processed material(straight through processing) and material stored at 2-8° C. for up tothree days. See FIG. 16 (VCC) and FIG. 33 (RM). The data show that bulkdrug substance (BDS) may be stored for three days at 2-8° C. beforeprocessing and still achieve acceptable results post-lyophilization.

Conclusion.

This study demonstrates that a 3 day hold of the drug substance at 2-8°C. can still result in acceptable long-term stability of the lyophilizeddrug product allowing for adding flexibility during routinemanufacturing.

2.12. Presentation of Product.

2.12.1. WP7-Lyo14.

Materials and Methods.

The ADXS-HPV drug product was used for this study. The formulation wasphosphate-based with 2.5% sucrose, the residual moisture target was2.5-3.0%, and the OD₆₀₀=10. The stability conditions used were 2-8° C.,−20° C. and accelerated conditions for 1, 2, and 3 days at 30° C. Thefactors tested were 2R vials, 1×10⁹ VCC, and 1.2 mL fill.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, materialwas harvested and processed for lyophilization. The lyophilization runwas performed for a target residual moisture of 2.5-3.0%. VCC wasanalyzed before and after lyophilization and at accelerated conditions.VCC titer was measured, expressed both as count and percent of countbefore lyophilization.

Results.

The data show a decrease in viability under accelerated conditions butstill within specifications. The data also indicate 2R vial presentationis suitable for use in lyophilization using the described compositionsand methods. See FIG. 17 (VCC) and FIG. 34 (RM). The residual moisturewas ˜2% and below the target of 3-4%.

2.13 Batch Scale

Materials and Methods.

The ADXS-HPV drug product was used for this study. The formulation wasphosphate-based with 2.5% sucrose, the residual moisture target was2.5-3.0%, and the OD₆₀₀=10 with a target of 1×10¹⁰ CFU/mL. 2 mL ADXS-HPVdrug product were added to each of about 1500 R6 vials. The stabilityconditions used were 2-8° C., −20° C. and accelerated conditions for 1,2, and 3 days at 30° C.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, materialwas harvested and processed for lyophilization. The lyophilization runwas performed for a target residual moisture of 2.5-3.0%. VCC wasanalyzed before and after lyophilization and at accelerated conditions.VCC titer was measured, expressed both as count and percent of countbefore lyophilization. Residual moisture (RM) was also determined.

Results.

The described compositions and methods are suitable for use with scalebatch lyophilization. Accelerated stability is consistent withdevelopment of scale batches. Batch scale demonstrated suitability forclinical supply of drug substance in 6R vial presentation. See FIGS.35-36 (VCC), FIGS. 37-38 (RM), and Table 12. “H” and “C” in FIG. 37refer to hot and cold snots respectively within the lyophilizer.

TABLE 12 Raw Data Test Test Method Reference Result Visual LAB-GEN-810LAB-GEN-810- 20 vials with appearance rev. 1.0 TR01: T17L004 white tooff white cake 20 vials with white to off white solution pH LAB-GEN-840LAB-GEN-840- pH: 6.77 rev. 1.0 TR01: T17L007 Osmolality LAB-GEN-800LAB-GEN-800- 372 mOsm/kg rev. 1.0 TR01: T17L008 Extractable LAB-GEN-820LAB-GEN-820- 1.92 mL per vial volume Rev. 1.0 TR01: T17L005 CCITLAB-GEN-830 LAB-GEN-830- No ingress of rev. 1.0 TR01: T17L006 dye in 10vials Viable cell TM-SB-532 TM-SB-532- 6.4 × 10⁹ CFU/mL count Rev. 2.0TR01: T17L0172.14. Exemplary Materials and Methods.

pH-Buffer Stock Solutions.

pH-buffer solutions were prepared as 4-fold stock solutions.

Phosphate Buffer Stock Solution.

The phosphate buffer stock solution was prepared to resemble the currentdrug substance formulation as closely as possible, however without NaCland KCl. It was prepared as a 4-fold stock: 0.8 g of KH₂PO₄ (anhydrous)and 4.6 g of Na₂HPO₄ (anhydrous) were dissolved in 800 mL of WFI. pH wasadjusted to 7.2 with 10% HCl. Solution was filled up to 1000 mL of WFIand was filtered sterile with a 0.2 μm filter. The final formulated drugproduct would contain 0.2 g/L KH₂PO₄ (anhydrous) and 1.15 g/L of Na₂HPO₄(anhydrous).

Citrate Buffer Stock Solution.

A 40 mM (=4-fold) stock solution of citric acid was prepared bydissolving 0.84 g of citric acid ad 100 mL WFI. A 40 mM (=4-fold) stocksolution of Na-citrate was prepared by dissolving 2.94 g of Na-citratead 250 mL WFI. A 40 mM (=4-fold) stock solution of citrate buffer wasprepared by adjusting the pH of the 40 mM Na-citrate solution to pH=7.2using the 40 mM citric acid solution. Solution was filtered sterile witha 0.2 μm filter.

MOPS-Buffer Stock Solution.

A 40 mM (=4-fold) stock solution of MOPS was prepared by dissolving2.3125 g MOPS in 200 mL of WFI. pH was adjusted to pH=7.2 with 10% HCl.Solution was filled up ad 250 mL WFI and was filtered sterile with a 0.2μm filter.

40% w/v Sucrose Stock Solution.

200 g of sucrose were dissolved ad 500 mL WFI. Solution was filteredsterile with a 0.2 μm filter.

40% w/v Trehalose Stock Solution.

40 g of trehalose were dissolved ad 100 mL WFI. Solution was filteredsterile with a 0.2 μm filter.

20% w/v MSG Stock Solution.

8 g of L-Glutamic acid monosodium salt hydrate were dissolved ad 40 mLWFI. Solution was filtered sterile with a 0.2 μm filter.

20% w/v rHSA Stock Solution.

10 g of lyophilized rHSA (Kerry, rAlbumin EG) were dissolved ad 50 mLWFI. Solution was filtered sterile with a 0.2 μm filter.

Buffers for Resuspension of the Bacterial Pellet.

1-fold buffers for resuspension of the bacterial pellet were prepared bymixing 140 mL WFI, 10 mL of 40% sucrose and 50 mL of the respective4-fold pH-buffer stock resulting in 1×pH buffer/2% sucrose.

AA-Mix Stock Solution.

200 mL of a 4-fold amino acid stock solution were prepared thatcontained 144 mM arginine, 228 mM glutamine and 28 mM isoleucine. See,e.g., Paik et. al. (Biotechnol Prog. 2012 November-December; 28(6),herein incorporated by reference in its entirety for all purposes).Final amino acid concentrations in formulated drug substance were 36 mM,57 mM and 7 mM, respectively. It was prepared as follows: 5.17 g ofarginine (Mw=174.2 g/mol) were weighed and dissolved ad 50 mL WFI. 48.52mL (representing 5.017 g) of this solution were transferred into a 250mL bottle. 0.8 g of isoleucine (Mw=131.17 g/mol) were weighed anddissolved ad 50 mL WFI. 45.91 mL of this solution (representing 0.735 g)were transferred into the same 250 mL bottle. 6.71 g of glutamine(Mw=147.13 g/mol) were weighed and transferred directly into the 250 mLbottle. Total volume was filled up to 160 mL WFI and the pH wascarefully adjusted to pH=7.2 by adding 2N NaOH under constant stirring.When all glutamine was dissolved and the pH was stable, volume wasfilled up to 200 mL WFI. Solution was filtered sterile with a 0.2 μmfilter.

Preparation of 2-Fold Excipient Stocks.

2-fold excipient stocks were used for mixing 1:1 with the bacterialstock solution to obtain the final formulations with an OD₆₀₀˜10 orOD₆₀₀˜2.0. They were prepared by mixing appropriate volumes of 4-foldpH-buffer stock solutions and excipient stock solutions to obtain thedesired concentrations.

Cultivation of Lm.

100 mL of TSB (30 g TSB/1 kg WFI, 5 g yeast extract/1 kg WFI, additional7.5 g glucose/1 kg WFI giving a final concentration of glucose of 10 g/1kg WFI) were pre-warmed in a 500 mL baffled shake flask to 37° C. Mediumwas incubated overnight. On the next day, the medium was clear. One vialof the Lm RCB (5277-2015-01.01; VCC=2.44×10⁹ CFU/mL) was thawed at roomtemperature. Medium was inoculated with 900 μL of vial content. Lm wereincubated at 37° C., 110 rpm for 5 h 15 minutes (P1). At this point theOD₆₀₀ was 3.59. For P2, 5×3 L Fernbach bottles containing 500 mL of TSBwere inoculated with 2.5 mL of P1, respectively. The cultures wereincubated at 37° C., 110 rpm for 14 h 50 min. Cultures were pooled andOD₆₀₀ was 5.96.

Alternatively, 50 mL of TSB were pre-warmed in a 250 mL baffled shakeflask to 37° C. Medium was incubated overnight. On the next day, themedium was clear. One vial of the Lm RCB (5277-2015-01.01; VCC=2.44×10⁹CFU/mL) was thawed at room temperature. Medium was inoculated with 600μL of vial content. Lm were incubated at 37° C., 110 rpm for 7 h 55 min(P1). At this point the OD₆₀₀ was 4.78. For P2 3×3 L Fernbach bottlescontaining 500 mL of TSB were inoculated with 5.0 mL of P1,respectively. The cultures were incubated at 37° C., 110 rpm for 14 h 30min. At this point the OD₆₀₀ was 5.96.

Concentration and Formulation of Lm.

For each pH-buffer (phosphate, citrate & MOPS) 520 mL of P2 werecentrifuged at 2,000 g, 10 min, RT. Supernatant was discarded and thepellets were each resuspended in 155 mL of 1×pH-buffer 2% sucrose,respectively and the OD₆₀₀ values checked. Target OD₆₀₀ was 20 afterconcentration: OD₆₀₀ phosphate buffer=19.2. OD₆₀₀ citrate buffer=18.6.OD₆₀₀ MOPS buffer=18.8. Final formulations were obtained by mixing equalvolumes of concentrated bacteria with 2×-concentrated excipient stocksresulting in a formulation with an OD₆₀₀˜10. Excipient concentrationsare described elsewhere herein. After formulation a sample was taken forVCC analysis (before Lyo).

Alternatively, 1,000 mL of P2 were centrifuged at 2,000 g, 10 min, RT.Supernatant was discarded and the pellets were each resuspended in 300mL of 1× phosphate buffer 2% sucrose. OD₆₀₀ was 18.3 after resuspension.This stock was used for generation of the samples of OD₆₀₀˜10. Forgeneration of the samples of OD₆₀₀˜2 this stock was diluted by a factorof 4.58 with 1× phosphate buffer 2% sucrose. OD₆₀₀ was 4.12 afterdilution. Final formulations were obtained by mixing equal volumes ofconcentrated bacteria with 2× concentrated excipient stocks resulting informulations with an OD₆₀₀˜10 or OD₆₀₀˜2.

2.15. Summary.

In summary, the data show that accelerated conditions appear to be agood predictor of long-term stability and that storage of lyophilizeddrug product at 2-8° C. and −20° C. is possible. The data for ADVX-HPVare comparable to the data for ADXS-HER2, indicating that the data ispredicted to be consistent across different Lm drug products. The dataalso indicate that higher RM is more desirable. For example, moisturesbelow about 1% may not provide a stable Lm product, but moistures ashigh as 6-7% appear to be as stable as moistures around 3-4%. The dataalso indicate that the temperature shift improves stability, and thathigher shelf temperature (primary drying step) improves stability. 2Rand 6R vial presentation is suitable for use in lyophilization using thedescribed compositions and methods. Bioassay shows good activity oflyophilized material compared to frozen liquid formulation. Batch scaleis suitable clinical supply in a 6R presentation. In some embodiments,the drug product is presented in 6R vials at 1×10¹⁰ CFU/mL. In someembodiments, the drug product is presented in 2R vials at 1×10⁹ CFU/mL.

Induction of a stress response through a temperature shift significantlyimproves the viability after lyophilization. In addition, despitephosphate buffers generally not being ideal buffers for lyophilizationproducts, phosphate-based formulation had better performance compared tocitrate- and MOPS-based buffers and required the smallest process changeas they were closest to the current drug substance formulation. The beststability was seen in formulations including sucrose but no trehalose,MSG, or rHSA. Formulations containing sucrose-only or a mixture ofsucrose+AA had better residual moisture compared to rHSA formulations,which were drier. Highest recoveries (and lowest variability) wereobserved at lowest sucrose concentration (about 2.5% w/v). Improvementin stability was observed with increased shelf temperature during theprimary drying step (e.g., about 18° C.) and with increased residualmoisture levels (e.g., about 3.5%). For increased residual moisturelevels such as 3.5% (which are higher than typical residual moisturelevels for lyophilized products), secondary drying temperatures as lowas 5° C. may be feasible (e.g., between about 5° C. and about 20° C.).

Example 3. Reproduction and Further Optimization of LyophilizationParameters for Listeria monocytogenes

A series of experiments were performed with different test parameters toreproduce lyophilization cycles from Example 2 and to further optimizethe formulation, the pre-conditioning of cells, and the lyophilizationcycle.

3.1. Reproduce Previous Lyophilization Cycle as Basis of Comparison.

A series of experiments (WP2A) were performed with 6R vials and 2 mLfills using the same lyophilization cycle parameters as Lyo9 throughLyo13.

Study Design.

Once the material in the bioreactor reached the target OD₆₀₀, materialwas harvested and processed for lyophilization. The lyophilization runwas performed for a target residual moisture of 2.5-3.0%. VCC wasanalyzed before and after lyophilization and at accelerated condition(1, 2, and 3 days at 30° C.). Residual moisture was also measured afterlyophilization and at the 3 days accelerated condition. Micro-FlowImaging (MFI) and Resonant Mass Measurement (RMM) were also performedbefore lyophilization, after lyophilization, and at acceleratedconditions for 1, 2, and 3 days (30° C.). VCC titer was measured,expressed as both count and percent of count before lyophilization.Moisture and VCC data were compared to previous data from the samelyophilization cycle conditions.

Results.

A decrease of the VCC (CFU/mL) to 80% was observed after lyophilization.See FIG. 18. The VCC was constant throughout storage for up to 72 hoursat 30° C. Initial residual moistures (direct sample injection) averaged2.4%, and no increase was observed over 72 hours at 30° C. The number ofsubvisible particles of samples before and after lyophilization wascomparable as measured by MFI and RMM. The particle size distributionremained constant. The previous lyophilization cycle was successfullyreproduced.

3.2. Study Residual Moisture Content as Function of Secondary DryingShelf Temperature.

A series of experiments (WP2B) were performed to study residual moisture(RM) content as a function of secondary drying shelf temperature topredict secondary drying shelf temperatures that would result in thetarget of 3.5% RM.

Study Design.

6R vials with 2 mL fills and the same freezing and primary drying as inthe WP2A experiments. Secondary drying was conducted in stages: (1) endof primary drying, stopper shelf (for RM information only); (2) ramp to0° C., hold for 6 hr, stopper shelf; (3) ramp to 5° C., hold for 6 hr,stopper shelf; (4) ramp to 15° C., hold for 6 hr, stopper shelf. VCC wasanalyzed before and after lyophilization and at accelerated condition(1, 2, and 3 days at 30° C.). Residual moisture was also measured afterlyophilization and at the 3 days accelerated condition. MFI and RMM werealso performed before lyophilization, after lyophilization, and ataccelerated conditions for 1, 2, and 3 days (30° C.).

Results.

The target RM of 3.5% can be obtained by using a secondary dryingtemperature between 5° C. and 15° C. (e.g., 12° C.). See FIG. 19. Thenumber of subvisible particles of samples at different sampling pointswas comparable as measured by MFI and RMM. Different SD temperatures hadno influence on the subvisible particle level. Comparable particlelevels were observed for WP2A samples. No significant change in VCC wasobserved from pre-lyophilization to post-lyophilization. A decrease ofthe VCC to 78%-85% of pre-lyophilization was observed after 72 hr at 30°C. There was no discernible trend of VCC with SD temperatures.

3.3. Evaluate Modified Freezing Steps.

A series of experiments (WP3) were performed to explore a modifiedfreezing step with an extended hold time of vials at −4° C. to allow allvials to equilibrate just above freezing temperature.

Study Design.

6R vials with 2 mL fills and the cycle conditions from the WP2Aexperiments were used with the following changes: (1) extend hold at −4°C. from 30 min to approximately 1 hr 20 min; and (2) conduct secondarydrying at 12° C. to target 3.5% RM. VCC was analyzed before and afterlyophilization and at accelerated condition (1, 2, and 3 days at 30°C.). Residual moisture was also measured after lyophilization and at the3 days accelerated condition. MFI and RMM were also performed beforelyophilization, after lyophilization, and at accelerated conditions for1, 2, and 3 days (30° C.).

Results.

Primary drying (PD) of samples was competed after about 23 hours processtime. For front vials, PD was already finished after 18 hours. For backvials, PD was finished after 20 hours. After PD, the vials wereequilibrated for 83 min at −4° C. After lyophilization, samples wereanalyzed immediately (Tlyo) or stored at 30° C. (Txxh). A decrease ofthe VCC to 80% was observed after lyophilization and to 70% afterstorage for up to 72 hr at 30° C. (T30 h). A RM of 2.3% was measuredafter lyophilization for the center vials instead of the expected 3.5%.The number of subvisible particles of the samples before and afterlyophilization was comparable as measured by MFI. The particle sizedistribution remained constant. The enhanced freezing step may havechanged the ice crystal formulation and thereby the drying behavior ofthe lyophilization cake, which may have influenced the cell viabilityand residual moisture. See FIGS. 40A-B.

3.4. Evaluate Modified Freezing Steps and Primary Drying Temperatures.

A two-factorial design study (WP4) is performed (freezing step andprimary drying temperature).

Study Design.

Fast shelf cooling from 5° C. to −45° C. during the freezing step istested. Adjusted primary drying conditions are tested to reduce cakeshrinkage/collapse. VCC is analyzed before and after lyophilization andat accelerated condition (1, 2, and 3 days at 30° C.). Residual moistureis also measured after lyophilization and at the 3 days acceleratedcondition. MFI and RMM are also performed before lyophilization, afterlyophilization, and at accelerated conditions for 1, 2, and 3 days (30°C.).

3.5. Evaluate Thawing Procedures (37° C. Thaw).

A series of experiments (WP6) were performed to evaluate a new thawingprocedure for ADXS-HER2. The previous thawing procedure was to thawformulated bulk material overnight at 2-8° C.

Study Design.

6R vials with 2 mL fills and the cycle conditions from the WP2Bexperiments were used. Formulated bulk material at an OD₆₀₀ of 10 wasthawed at 37° C. The cell pellet was thawed at 37° C. and then dilutedwith formulation buffer to an OD₆₀₀ of 10. VCC was analyzed before andafter lyophilization and at accelerated condition (1, 2, and 3 days at30° C.). Residual moisture was also measured after lyophilization and atthe 3 days accelerated condition. MFI and RMM were also performed beforelyophilization, after lyophilization, and at accelerated conditions for1, 2, and 3 days (30° C.). See FIGS. 41A-B.

Results.

The process of WP2B was reproduced until SD temperature of 5° C. Primarydrying (PD) of samples completed after ˜25 h process time. Lyo processwas comparable to WP2B. After lyophilization, samples were immediatelyanalyzed (Tlyo) or stored at 30° C. (Txxh). CFU/mL at Tliq werecomparable between A (formulated bulk material (i.e., Drug Substance))and B. (cell pellet (i.e., Drug Substance that has been highlyconcentrated to essentially remove all formulation buffer)). Afterlyophilization, a decrease to 70% and 80% VCC was observed. A furtherdecrease to about 50% VCC was observed after storage for 24 hours at 30°C., which was unchanged after 72 Hr. at 30° C.

3.6. Evaluate Different Bacterial Target Concentrations (WP7).

Three different bacterial target concentrations were tested to determinethe influence of the bacterial concentration on the cake appearance whenthe lyophilization cycle with an annealing step is used.

Study Design.

Three different formulations with different OD600 values were prepared:

(a) OD 10: F1000: use of delivered BDS as provided,

(b) OD 2: F0200: 31.37 ml BDS+118.63 ml formulation buffer, and

(c) OD 0.65: F0065: 10.20 ml BDS+139.80 ml formulation buffer.

HER2 material was provided using the ADXS platform manufacturing process(see Example 7).

Parameter Cell Pellet Amount 800 mL (by volume OD600 (raw material)10.05    Cells/mL 1.69 × 10¹⁰ Viability 98.45%VCC was analyzed before lyophilization, after lyophilization, and ataccelerated conditions for 1, 2 and 3 days (30° C.). Residual moisturewas analyzed after lyophilization and 3 days accelerated condition. MFIand RMM were analyzed before lyophilization, after lyophilization, andat accelerated conditions for 1, 2, and 3 days (30° C.).

Results:

Primary drying (PD) of samples was completed after ˜40 h process time(indicated by both Pt₁₀₀ sensor and pressure sensor readout). Theprocess was comparable to WP7 cycle 1; only the SD temperature waschanged from 5° C. to 0° C. to target a residual moisture content of ca.3.5%. After lyophilization, samples were immediately analyzed (Tlyo) orstored at 30° C. or 2-8° C., respectively. A correlation betweenbacterial concentration and optical appearance of the final product wasobserved. The lower the bacterial concentration, the more cake shrinkagewas observed. Reconstitution of the lyophilized cake was faster forF0065 and F0200 (˜20 s) than for F1000 (˜100 s). See FIGS. 42A-B.

After lyophilization, a decrease in VCC to about 60% was observed,independent of the bacterial concentration. Also after lyophilization,no difference in VCC was observed for front and center vials. A slightlyhigher VCC was even detected for front vials. After 24 h at 30° C. afurther 10% decrease in VCC was observed for the two lower bacterialconcentrations. Results were unchanged after 72 h. at 30° C. After 7days at 2-8° C., a decrease in VCC of about 20% was observed. See FIG.43.

Viable cell count (VCC) and viability appeared stable under acceleratedconditions. See FIGS. 44A-B.

3.7. Evaluate 2R Vial Presentation (WP8).

2R vial presentation at a target VCC of 1×10⁹ wash was evaluated. Inaddition, stability of frozen and non-frozen BDS was compared.

Study Design.

Formulated bulk material at a target VCC of 1×10⁹ and 1×10¹⁰ plus about30% to account for manufacturing losses was provided. VCC was analyzedbefore lyophilization, after lyophilization, and at acceleratedconditions for 1, 2, and 3 days (30° C.). Residual moisture was analyzedafter lyophilization and 3 days accelerated condition. MFI and RMM wereanalyzed before lyophilization, after lyophilization, and at acceleratedconditions for 1, 2, and 3 days (30° C.).

Results.

Minimal losses due to lyophilization were observed. No significantchanges observed in VCC or live/dead on accelerated stability wereobserved. % live at initial was higher relative to liquid-frozenformulation. See FIGS. 45A-B.

3.8 Large Scale Production (WP7).

In a large scale production the holding times of the BDS before startinga lyophilization run are longer compared to lyophilization runs duringdevelopment in a pilot scale freeze dryer. The aim of this study is toevaluate whether this could have an influence on the product.

Study Design.

Non-frozen liquid bulk drug substance (BDS) was provided by APC one dayprior to the start of the freeze drying cycle. The liquid BDS wasdiluted to an OD₆₀₀ value of 0.85 (target VCC of 1.3×10⁹ CFU/ml) byusing the delivered formulation buffer. The diluted material was storedat 2-8° C. during the conduct of the holding time study. The fourshelves were filled with BDS and loaded into the freeze-dryer at fourdifferent time points: 20 h (H20 h), 8 h (H8 h), 5 h (H5 h), and 0 h (H0h) before starting the freeze-drying process.

Results.

After lyophilization, a decrease in VCC was observed (relative to Tliq):78% at H0 h, 74% at H5 h: 74%, 73% at H8 h, and 65% at H20 h. Afterstorage for up to 72 h at 30° C. a further decrease of around 10% wasobserved for H20 h, H8 h, and H5 h. The VCC at T7 days after storage at2-8° C. was comparable to the VCC after lyophilization. See FIGS. 46A,46B, 47A, and 47B.

Example 4. Lyophilization of Drug Product and Long-Term Room TemperatureStability of the Lyophilized Drug Product

4.1 ADXS11-001 Pilot Batch

4.1.1 Materials and Methods

Previous development experiments have all been performed using asmall-scale development lyophilizer. Because scale up does not guaranteethat the same dynamics of product temperature and ice content as thoseat the laboratory scale modifications to the lyophilization cycle may beneeded for larger scale production. In order to evaluate potential scaleup issues a pilot batch was manufactured for proof of concept andstability studies. The Drug Substance process is carried out within asingle use closed system provided by rocking wave motion bioreactortechnology. The Drug Substance manufacturing followed the ADXS platformmanufacturing process. The platform consists of a single-use closedsystem of a product 20 L culture bag for fermentation, a tangential flowfiltration (TFF) manifold for concentration and buffer exchange and acontainer manifold for DS filling. The Drug Substance was held overnightat 2-8° C., diluted to the target OD₆₀₀ with formulation buffer, filledinto DIN 6R vials (2.0 mL) and lyophilized. The approximate batch sizewas 1500 vials.

TABLE 13 Primary Packaging Materials used for Pilot Batch. PrimaryPacking Material Specifications Supplier 6R Glass vials Rofa, 6R HALAllergy, MW005-002 Lyo stoppers West, 7000-5780 Adelphi, FDW20RTS Readyto sterilize (steam sterilization) Crimp cap, flip-off seals West,5921-2032 Adelphi, FOT20W The stoppers were delivered ready-to-processand were not dried before use.4.1.2 Study Design

The lyophilization process was conducted in a Martin Christ Epsilon2-12D pilot-scale lyophilizer. Because this lyophilizer uses a Piranigauge as the controlling pressure sensor instead of an MKS sensor usedin the laboratory-scale lyophilizer, a pressure set point for the Piranigauge had to be selected. Based on a review of previous lyophilizationcycles where the Pirani gauge pressure was measured (but not used forcontrol), a Pirani pressure of 0.163 mbar was found to be equivalent toa MKS pressure of 0.090 mbar during the main portion of primary drying.Since Pirani gauge pressure is dependent upon the composition of the gasphase, as the partial pressure of water decreases towards the end ofprimary drying, the Pirani pressure readings approach the MKS pressurereadings.

TABLE 14 Lyophilization Parameters for ADXS11-001 Pilot Batch Temp,T_(s) of Duration Shelves Vacuum Step Phase Item [hh:mm] [° C.] [mbar] 1Preparation Warm up of lyo, NA 4 off placement of temperature and Rxsensors 2 Load Loading of Shelves NA 4 off 3 Ramp Freezing Ramp samples00:20 −4 off to −4° C. (0.4° C./min) 4 Hold Hold/anneal samples 00:30 −4off to −4° C. 5 Ramp Freeze ramp to −45° C. 01:30 −45 off (0.45° C./min)6 Hold Hold at −45° C., 01:30 −45 off temperature equilibration acrossload 7 Hold Hold and preparation 00:30 — — of vacuum pump 8 Ramp PrimaryVacuum¹ 00:01 −45   0.160*** 9 Hold Drying Vacuum¹ 00:15 −45 0.160 10 Ramp Heating ramp 00:27 −18 0.160 (1.0° C./min) 11  Hold Stable shelf>tbc² −18 0.160 temp:primary drying 12  Ramp Secondary Heating ramp03:10 20 0.160 Drying (0.2° C./min) 13  Hold Stable shelf 02:00 20 0.160temp:secondary drying 14  Ramp- Hold Ramp to 10° C. 00:10 10 0.160 15 Hold Hold at 10° C. until NA 10 1.0³   unloading 15a Ventilation and NA10 500 mbar manual vial closure ¹Pirani vacuum sensor is processcontrolling (***0.163 was not programmable) ²End of primary drying isdefined here 14 hours after T_(P) P100 probe for the cold spot(s) havecrossed the T_(s) set point of −18° C. ³Only done to avoid vacuum pullafter the cycle has finished during the night4.1.3 Results and Discussion

TABLE 15 OD₆₀₀ and VCC for In-Process Samples Process Step OD₆₀₀ VCCWAVE Harvest 7.9 1.76 × 10¹⁰ Pre-Formulation 17 3.09 × 10¹⁰ Final BulkFormulation Post Hold/ 10.2 1.27 × 10¹⁰ Final Formulation FinalFormulated Bulk (before Lyo 10.2 1.76 × 10¹⁰

Preliminarily mapping of the lyophilizer was performed by determiningthe VCC (plate method) for hot (H) and cold (C) spots per shelf withinthe lyophilizer. The data are presented in FIG. 48 and Table 16.

TABLE 16 VCC and Residual Moisture Data from Hot and Cold Spots withinthe lyophilizer for the Pilot Batch Location Sample VCC (CFU/mL)Residual Moisture Hot 1 7.00E+09 2.57 2 7.60E+09 2.04 3 6.58E+09 2.39 46.94E+09 2.37 5 7.28E+09 2.16 Cold 1 9.78E+09 2.45 2 1.02E+10 2.53 31.01E+10 2.81 4 1.03E+10 2.61 5 1.12E+10 2.42

Summary statistics for the VCC data from the Hot and Cold spots withinthe lyophilizer demonstrate that the mean VCC for the Hot spots is7.08E+09 CFU/mL and the mean for the Cold spots is 1.032E+10 CFU/mL. Hotspots and cold spots in the lyophilizer are determined based ontemperatures from probes within the lyophilizer. Hot spots tend to be onthe edges of the lyophilizer, and cold spots tend to be in the center ofthe lyophilizer. The sample numbers correspond to the shelf within thelyophilizer and show little variation with the H or C locations. Referto the CV column in Table 17.

TABLE 17 Statistics Summary Coef Var Variable Location N Mean StDev (CV)VCC Cold 5 10316000000 531300292 5.15 (CFU/mL) C Hot 5 7080000000382883794 5.41

The data show a distinction in VCC and RM between the hot and cold Spotswithin the lyophilizer. It is not known if the difference in VCC isnecessarily due to the difference in RM, or whether they are bothfunctions of some other characteristic of the lyophilization cake.

Release and stability analysis was performed by Eurofins using validatedmethods. Vials are reconstituted with 2 mL or normal saline prior toanalysis. The release and stability data are provided in Table 18.

TABLE 18 Release and Stability Testing for ADXS11-001 Pilot Batch (Lot#5329PD-17-01) Results Release Accelerated stability Test Test methodSpecifications testing Day 1 Day 2 Day 3 Visual LAB-GEN-810 CakeWhite/off-white to appearance slightly yellow powder Color - White tooff-white 20 vials with 12 vials with 12 vials with 12 vials withReconstituted suspension white to off white to off white to off white tooff Solution white color white color white color white color Particles-Essentially free 20 vials with 12 vials with 12 vials with 12 vials withReconstituted of foreign particles no particles no particles noparticles no particles Solution present present present present pHLAB-GEN-840 6.0-8.0 6.77 6.79 6.80 6.79 Osmolality LAB-GEN-800 250-450mOsm/kg 372    — — — Extractable LAB-GEN-820   >1 mL/vial 1.92 mL — — —volume CCIT LAB-GEN-830 No Dye Ingress No ingress of dye — — — in 10vials Reconstitution LAB-GEN-880 Report Result (seconds) 56    24   28    27    time Viable cell TM-SG-110 5 × 10⁹- 1.12 × 10¹⁰ 1.16 × 10¹⁰1.09 × 10¹⁰ 1.10 × 10¹⁰ count 5 × 10¹⁰ CFU/mL % of viable TM-SG-110 >60%86%  89%  83%  87%  cells Endotoxin TM-SG-101 EU/mL <10 EU/mL — — —Monosepsis TM-SB-534 TYMC < 10 <1 CFU/L — — — CFU/mL TYMC: Absent in 1mL Absent in 1 mL — — — Candida albicans Absent in 1 mL Absent in 1 mLAbsent in 1 mL — — — Escherichia coli Bacillus subtilis Pseudomonasaeruginosa Staphylococcus aureus Clostridia sp. Absent in 10 mL Absentin 10 mL Absent in 10 mL — — — Salmonella sp. Single Species Pureculture Pure culture — — — (L. monocytogenes) Results T1 T3 T6 Test Testmethod 5° C. −20° C. 5° C. −20° C. 5° C. −20° C. Visual LAB-GEN-810 CakeIntact Intact appearance cake cake Color - 12 vials with 12 vials with12 vials with 12 vials with 12 vials with 12 vials with Reconstitutedwhite to off white to off white to off white to off white to off whiteto off Solution white color white color white color white color whitecolor white color Particles- 12 vials with 12 vials with 12 vials with12 vials with 12 vials with 12 vials with Reconstituted no particles noparticles no particles no particles no particles no particles Solutionpresent present present present present present pH LAB-GEN-840 6.80 6.816.79 6.79 6.87 6.86 Osmolality LAB-GEN-800 — — — — — — ExtractableLAB-GEN-820 — — — — — — volume CCIT LAB-GEN-830 — — — — — —Reconstitution LAB-GEN-880 20    22    21    21    36    29    timeViable cell TM-SG-110 1.24 × 10¹⁰ 1.22 × 10¹⁰ 1.15 × 10¹⁰ 1.11 × 10¹⁰1.15 × 10¹⁰ 1.21 × 10¹⁰ count % of viable TM-SG-110 88%  87%  88%  88% 88%  89%  cells Endotoxin TM-SG-101 — — — — — — Monosepsis TM-SB-534TYMC < 10 — — — — — — CFU/mL TYMC: — — — — — — Candida albicans Absentin 1 mL — — — — — — Escherichia coli Bacillus subtilis Pseudomonasaeruginosa Staphylococcus aureus Clostridia sp. Absent in 10 mL — — — —— — Salmonella sp. Single Species — — — — — — (L. monocytogenes)4.2.1 Accelerated Stability

Early development data demonstrated that accelerated stability at 30° C.may be predictive of long-term stability trends. The batch was stored at30° C. and evaluated for up to 63 days to determine how long the productwas stable for under accelerated conditions (FIGS. 49-53 and Table 19).

TABLE 19 VCC and % Live for ADXS11-001, Lot# 5329PD-17-01 Stored at 30°C. Day Specification % Live VCC (cells/mL) 0 ≥60% 86% 1.12 × 10¹⁰ 1 83%1.32 × 10¹⁰ 3 86% 1.35 × 10¹⁰ 7 89% 1.43 × 10¹⁰ 14 91% 1.09 × 10¹⁰ 2192% 1.51 × 10¹⁰ 23 88% 1.46 × 10¹⁰ 28 91% 1.01 × 10¹⁰ 35 90% 1.11 × 10¹⁰42 90% 1.08 × 10¹⁰4.2.2 In Vivo Testing

ADXS11-001 (AXAL) is a live attenuated Listeriamonocytogenes-listeriolysin O (Lm-LLO) immunotherapy that is underclinical development for the treatment of human papilloma virus(HPV)-associated cancers. ADXS11-001 is bioengineered to secrete anantigen-adjuvant fusion protein consisting of a truncated fragment oflisteriolysin O (tLLO) fused to the full length E7 protein of HPV 16(tLLO-E7). The proposed mechanism of action of the Lm-basedimmunotherapy is to stimulate both the innate and adaptive immunesystems in order to initiate a coordinated anti-tumor responseculminating in the de novo generation of tumor antigen-specific T cellsthat are capable of infiltrating and destroying the tumor. In order toconfirm that the bioactivity of the product is not adversely impacted bylyophilization tumor-bearing mice immunized with ADXS11-001 generateCD4+ and CD8+ T cells specific to HPV16-E7 and HPV16-E6.

The abilities of lyophilized AXAL and clinical AXAL to control tumorsand to prolong animal survival were evaluated and compared in TC-1tumor-bearing mice. Adult female C57BL/6 mice were injectedsubcutaneously in the right flank with 1×10⁵ TC-1 tumor cells and thenimmunized on Days 8, 15, and 22 after tumor implantation by IP injectionwith PBS or with various doses (5×10⁷ CFU, 1×10⁸ CFU, 2×10⁸ CFU) oflyophilized AXAL or clinical AXAL (see FIG. 54). Tumor growth and thegeneral health of the mice were monitored for 62 days after tumorimplantation. Mice were euthanized if tumor volume exceeded 2000 mm³.

In mice treated with PBS, tumor volume continued to increase, and noanimals survived past Day 30 (FIGS. 55-56). In comparison, all doses oflyophilized AXAL and clinical AXAL significantly inhibited tumor growthand prolonged animal survival (FIGS. 55-56). Notably, for each dose, thetumor growth curves and survival curves for lyophilized AXAL andclinical AXAL were similar.

As shown in FIG. 55, tumor-bearing mice were treated on day 8 post-tumorimplantation with PBS or with 3 different doses of lyophilized AXAL orclinical AXAL and at 7-day intervals thereafter for a total of 3 doses.Tumor volume was measured twice a week. Tumor growth curves for eachdose group are shown. ****P<0.0001. NS, not significant.

As shown in FIG. 56, tumor-bearing mice were treated on day 8 post-tumorimplantation with PBS or with 3 different doses of lyophilized AXAL orclinical AXAL and at 7-day intervals thereafter for a total of 3 doses.Tumor growth and the general health of the mice were monitored for 62days after tumor implantation. Mice were euthanized if tumor volumeexceeded 2000 mm³. Survival curves for each dose group are shown.**P<0.01. NS, not significant.

No significant differences were observed between lyophilized AXAL andclinical AXAL in their abilities to control tumor growth and to prolonganimal survival in TC-1 tumor-bearing mice. These data indicate that thelyophilization process does not affect the antitumor activities of AXAL.

4.3 Conclusion

The pilot batch successfully demonstrated the application of the ADXS DSplatform manufacturing process to support a lyophilized Drug Product.The % live and VCC on accelerated stability is consistent with previousdevelopment studies.

Example 5. Ability to Freeze/Thaw the Drug Substance and ObtainComparable Results to Continuously Processed Material

5.1 WP7, Cycle 3

Different storage conditions of Drug Substance (frozen vs. non-frozen2-8° C.) were evaluated to see if the improvements in the lyophilizationcycle result in improved viability post lyophilization for DS that hasbeen through a single freeze-thaw.

5.2 Materials and Methods

Frozen (A) and non-frozen liquid (B) BDS were provided by APC (Dublin,Ireland) one day prior to the freeze-drying cycle. Approximately 800 mLof both 2-8° C. and Frozen DS was formulated to an approximately OD₆₀₀of 14 in 1 L LDPE bags. The frozen material was thawed in a water bathat 37° C. until the material contained no long contained ice crystals(thawing time: 2.5 h). The two Drug Substances (A and B) were diluted totwo different OD₆₀₀ values by using the formulation buffer (prepared atCoriolis). Table 20 gives an overview of the prepared formulations andthe target OD₆₀₀ values.

TABLE 20 Formulations and Target OD₆₀₀ values Formulation BDS OD₆₀₀Value A0085 Frozen Drug Substance 0.85 A1300 Frozen Drug Substance 13B0085 Non-Frozen Drug Substance 0.85 B1300 Non-Frozen Drug Substance 13

The measured OD₆₀₀ values of the Drug Substance as well as of thedilutions are shown in Table 21. The dilution scheme for the preparedformulation is given in Table 22.

TABLE 21 Measured OD₆₀₀ Values of the Delivered Material. OD₆₀₀ valuesA: frozen B: non-frozen BDS (1000 mL, by volume) material materialAccording to shipping documents 13.8 13.8 Measured at Coriolis, n = 312.14 ± 0.12 11.33 ± 0.18 Formulations OD₆₀₀ Values A0085  0.86 ± 0.00A1300 12.14 ± 0.12 B0085  0.87 ± 0.00 B1300 11.33 ± 0.18

TABLE 22 Dilution Scheme for the Three Formulations - F1000, F0200, andF0065. Formulation DS Formulation Buffer A0085  14.00 mL 186.00 mL A1300200.00 mL — B0085  15.01 mL 184.99 mL B1300 200.00 mL —5.3 Study Design

Freezing during lyophilization was performed without an annealing stepor hold at 4° C., since no positive effect on the cake appearance wasobserved with those steps in previous experiments. The vials wereimmediately frozen to −45° C. without a holding or annealing step. Thesecondary drying time was prolonged to 5 hours to obtain a morehomogeneous batch and to reach the target residual moisture content (RM)of 3.5%. RM, VCC, MFI, RMM, reconstitution time, classification of theappearance of the lyophilized products, and determination of the waterloss by weighing was performed after lyophilization. VCC, MFI, RMM wereanalyzed at 30° C. for 24 and 72 h. Additionally, VCC was analyzed forsamples at 2-8° C. for 7 days.

TABLE 23 Lyophilization Cycle Parameters for WP7, Cycle3 TotalTemperature Pressure* Time Ramp Step Time [° C.] [mbar] [h] [K/min]loading  00:00** 4 1000 0.0 freezing 00:49 −45 1000 0.8 1.00 freezing02:00 −45 1000 2.8 Primary drying 00:30 −45 0.09 3.3 Primary drying00:15 −45 0.09 3.6 Primary drying 00:27 −18 0.09 4.0 1.00 Primary drying32:00:00 −18 0.09 36.0 Secondary 01:30 0 0.09 37.5 0.20 drying Secondary05:00 0 0.09 42.5 drying hold aeration 00:30 0 500 43.0 stoppering 00:01** 0 500 43.0 storage  00:30** 5 1000 43.1 0.50 *Pirani gaugecontrolling5.4 Results and Discussion

Lyophilization was performed using an Epsilon 2-12D pilot scalefreeze-dryer (Martin Christ, Osterode, Germany). During thefreeze-drying process pressure (by Pirani and MKS), product temperature,shelf temperature, and ice condenser temperature were monitored. Centerand front vials were monitored by PT₁₀₀ sensors.

No annealing step was included during freezing and the secondary dryingstep at 0° C. was set to 5 h. The primary drying of all samples wascompleted after about 34 h process time, as indicated by PT₁₀₀ sensorsand pressure sensor readout. For front vials the primary drying wasalready finished after 18 h process time as indicated by PT₁₀₀ sensors.The drying of the samples was independent of the bacterialconcentration.

For the freezing step, the samples were immediately frozen to −45° C.without a holding or annealing step. The front vials did not reach −45°C. before ramping to the PD temperature, according to the PT₁₀₀ sensors.

A video was recorded from the lyophilization process to determine whenthe shrinkage takes place. In the video, samples from all fourformulations (A0085, A1300, B0085 and B1300) were visible and theshrinkage seems to occur during primary drying. The drying behavior ofthe center vials might be a slightly different as primary drying wasfinished earlier for front vials than for center vials.

5.5 Optical Evaluation of the Freeze-Dried Product

The optical appearance of the lyophilized cakes was documented for tencenter vials of each formulation after lyophilization. The overalloptical appearance of the lyophilization cakes of the four formulationswas good. All cakes were compact and had no full contact to the glassvial. For the higher concentrated formulations (A1300 and B1300) theshrinkage in cake height and from the vial wall and bottom wascomparable to that of samples of the previous cycle with similar OD₆₀₀values (F1000). The same is true for the lower concentrated samples(A0085 and B0085). A similar lyophilization cake was observed as for thelower concentrated formulations of Cycle 2 (F0065). The lyophilizationcake had a similar optical appearance as the lyophilized placebo. Aswith cycle 2, a correlation between bacterial concentration and opticalappearance of the final product was observed.

5.6 Determination of the Cake Weight

The cake weight and the water loss were determined during cycle 3. Theweight of five vials per formulation was gravimetrically determined.Based on the weight of the empty vial, the vial after filling and afterlyophilization, the cake weight and the water loss were calculated(Table 24). The lyophilization cake of the lower concentratedformulations weighed 30 mg and that of the higher concentratedformulations 40 mg. After lyophilization, a water loss of 1.17-1.18 gwas determined. Therefore, a reconstitution volume of 1.2 mL is suitableto obtain the same bacterial concentration as prior to lyophilization.

TABLE 24 Determination of the Cake Weight and the Water Loss. Vial afterEmpty Vial Filled Vial Lyophilization Cake Weight Water Loss Water LossSample (g) (g) (g) (g) (g) (%) A1300 1 5.20 6.41 5.24 0.04 1.17 82 25.27 6.48 5.30 0.04 1.18 82 3 5.23 6.44 5.27 0.04 1.18 82 4 5.16 6.375.20 0.04 1.17 82 5 5.17 6.38 5.21 0.04 1.17 82 A0085 1 5.18 6.39 5.210.03 1.18 82 2 5.21 6.41 5.24 0.03 1.17 82 3 5.15 6.36 5.18 0.03 1.18 814 5.22 6.43 5.25 0.03 1.18 82 5 5.21 6.41 5.24 0.03 1.18 82 B1300 1 5.286.49 5.31 0.04 1.17 82 2 5.27 6.48 5.31 0.04 1.17 82 3 5.21 6.42 5.250.04 1.17 82 4 5.18 6.38 5.21 0.04 1.17 82 5 5.21 6.42 5.24 0.04 1.17 82B0085 1 5.26 6.46 5.29 0.03 1.18 82 2 5.25 6.47 5.29 0.03 1.18 82 3 5.266.47 5.29 0.03 1.18 82 4 5.23 6.44 5.26 0.03 1.18 82 5 5.27 6.48 5.300.03 1.18 825.7 Reconstitution Time

The reconstitution time was measured for two samples per formulation andcompared to the results of cycle 1 and cycle 2 (FIG. 57 and Table 25).The reconstitution time of the higher concentrated formulations (A1300and B1300) was longer than for the lower concentrated formulations(A0085 and B0085). The reconstitution times were, in general, shorterthan for the previous cycles of WP7. A direct freezing of the sampleswithout including an annealing step seems to shorten the reconstitutiontimes.

TABLE 25 Overview of the Measured Reconstitution Times of WP7 Cycle 1 toCycle 3. Reconstitution time Time (sec) Sample Mean SD Foam LP Cycle1_SD start_1 90 95 5 — yes Cycle 1_SD start_2 100 — yes Cycle 1_SD 2h_175 73 2 — yes Cycle 1_SD 2h_2 71 — no Cycle 1_SD end_1 77 69 8.5 — noCycle 1_SD end_2 60 — no Cycle 2 F0065_2 24 22 2 — no Cycle 2 F0065_3 20— no Cycle 2 F0200_1 21 21 0.5 — no Cycle 2 F0200_2 20 — yes Cycle 2F1000_1 107 104 3.5 — no Cycle 2 F1000_2 100 — yes Cycle 3 A0085_1 13 121 — no Cycle 3 A0085_2 11 — no Cycle 3 A1300_1 47 53 6 — no Cycle 3A1300_2 59 — yes Cycle 3 B0085_1 13 14 1 — no Cycle 3 B0085_2 15 — noCycle 3 B1300_1 72 63 9.5 — no Cycle 3 B1300_2 53 — no Score Foam: 0—nofoam, 1 slight foam, 2—moderate foam, 3—strong foam, 4—strong and steadyfoam. LP—low pressure.5.8 Micro-Flow Imaging (MFI)

The number of subvisible particles was analyzed by MFI to determinewhether the raw material (frozen or non-frozen) or the bacterialconcentration has an influence on particle formation as well as on thesize distribution of the particles. Subvisible particles are particulatematter that is not observable to the naked eye. Particulate matter ininjections and parenteral infusions consists of mobile undissolvedparticles, other than gas bubbles, unintentionally present in thesolutions. There are regulatory limits on the number of subvisibleparticles allowed in parenteral infusions. Samples were analyzed beforelyophilization (Tliq), after lyophilization (Tlyo), and after storagefor 24 hours and 72 hours at 30° C. (T24 h and T72 h). The results areshown in FIGS. 58A-D. The number of subvisible particles was unchangedfor the frozen material (A0085 and A1300) before and afterlyophilization as well as after storage at 30° C. for up to 72 h. Forthe non-frozen material, more particles were detected beforelyophilization. Similar results were obtained in other experiments wherenon-frozen material was also used. The number of subvisible particlesafter lyophilization was comparable to the results of the frozenmaterial.

5.9 Resonant Mass Measurement (Archimedes)

Results for RMM analysis of ADXS-HER2 samples regarding their content ofnegatively and positively buoyant particles are presented in FIGS.59A-D.

The results for the frozen and the non-frozen material were similarComparison of cumulative negatively buoyant particle counts for all timepoints and all storage conditions for bin size ≥0.3 μm. Note thatparticle counts below 300,000 particles per mL (LoQ), are given forinformation only (data not dilution-corrected, A0085 and B0085: 200-folddilution, A1300 and B1300: 5,000-fold dilution).

After lyophilization a smaller second particle population appeared ataround 300 nm. The particle distribution was comparable for allformulations throughout all analyzed time points. Comparison of thedifferential particle counts of negatively buoyant particles. Valuesbelow 0.3 μm size bins are given for information only (data notdilution-corrected, A0085 and B0085: 200-fold dilution, A1300 and B1300:5,000-fold dilution).

Because of the different bacterial concentrations of the formulations,different dilutions had to be prepared. A0085 and B0085 were 200-folddiluted and A1300 and B1300 were diluted 5,000-fold. The cumulativecounts are not corrected for the individual dilution, in order not tooverestimate the multiplication error inherent to the measurement.Furthermore, since the Limit of Quantitation (LoQ) of the method isapproximately 300,000 particles per mL, a dilution correction wouldelevate low particle counts for some measurements above the LoQ, whichwould otherwise not have been considered and might therefore not reflectthe actual experimental conditions.

The number of submicron particles was unchanged for all fourformulations before and after lyophilization (FIGS. 59A-D). The resultsfor the frozen and the non-frozen material were similar Beforelyophilization one main particle population was detected in the sizerange of 600-700 nm (FIGS. 60A-D). After lyophilization, a smallersecond particle population appeared at around 300 nm. The particledistribution was comparable for all formulations throughout all analyzedtime points.

5.10 Karl Fischer Titration

The RM at Tlyo was analyzed by direct injection (FIG. 61 and Table 26).A RM of about 3% was reached for the higher concentrated samples (A1300and B1300) and a RM of about 3.5% for the lower concentrated samples(A0085 and B0085). The relative standard deviation of the five analyzedvials per formulation was lower than for cycle 2, which is most likelydue to the extended SD time. The results of the frozen and non-frozenmaterial were comparable.

TABLE 26 Overview of the KF Results Measured by Direct Injection for WP7Cycle 3. RMC [%] Sample A0085 A1300 B0085 B1300 Vial 1 3.4 3.1 3.3 3.3Vial 2 3.1 3.0 3.5 3.4 Vial 3 3.3 2.8 3.5 3.2 Vial 4 3.7 3.2 4.1 2.7Vial 5 3.2 2.8 3.9 2.5 Mean 3.4 3.0 3.7 3.0 SD 0.2 0.2 0.3 0.35.11 VCC Assay

The concentration of viable bacteria (VCC, expressed as CFU/mL) wasanalyzed at Tliq, after lyophilization at Tlyo, after storage for 24 hand 72 h at 30° C. (T24 h and T72 h) and after storage for 7 days at2-8° C. (T7 days) (FIG. 62 and Table 27). After lyophilization, adecrease in VCC to about 60% (relative to Tliq) for the lower bacterialconcentration and to 70-78% for the higher bacterial concentration wasobserved. A further decrease of about 10% was observed for the lowerbacterial concentration and of 20% for the higher bacterialconcentration after an incubation time of 72 h at 30° C. The VCC resultsafter storage for 7 days at 2-8° C. were comparable to the values afterlyophilization. The results for the CFU/mL were, as in WP2, below thetarget values post-lyophilization:

A1300 (target OD₆₀₀ of 13): 1E+10 CFU/mL

A0085 (target OD₆₀₀ of 0.85): 1E+09 CFU/mL

B1300 (target OD₆₀₀ of 13): 1E+10 CFU/mL

B0085 (target OD₆₀₀ of 0.85): 1E+09 CFU/mL

APC (Ireland, Dublin) performed flow cytometry again with samples ofeach formulation. The VCC and % live results are presented in FIG. 62and Table 27.

TABLE 27 Overview of the CFU/mL and the Relative Viability. SampleCFU/mL SD % A0085 Tliq 5.67E+08 1.21E+08 100 Tlyo 3.17E+08 1.56E+07 56T24h 2.48E+08 2.00E+07 44 T72h 2.43E+08 1.26E+07 43 T7days 2.94E+082.06E+07 52 A1300 Tliq 5.63E+09 1.43E+09 100 Tlyo 4.40E+09 3.83E+08 78T24h 3.40E+09 2.08E+08 60 T72h 3.39E+09 1.55E+08 60 T7days 3.61E+092.56E+08 64 B0085 Tliq 5.03E+08 8.06E+07 100 Tlyo 3.41E+08 2.19E+07 68T24h 2.84E+08 1.89E+07 56 T72h 2.69E+08 2.24E+07 54 T7days 3.15E+081.45E+07 63 B1300 Tliq 6.23E+09 6.94E+08 100 Tlyo 4.37E+09 3.14E+08 70T24h 3.24E+09 2.76E+08 52 T72h 3.20E+09 1.33E+08 51 T7days 3.91E+091.72E+08 63

FIGS. 63A-B show the VCC and % live after Cycle 3 and on acceleratedstability.

Minimal losses were observed due to lyophilization. No changes wereobserved in VCC or % live on accelerated stability. % live at initial ishigher relative to the liquid-frozen formulation, which supports thefurther development of a lyophilized formulation. DS that had undergonea freeze/thaw demonstrated good accelerated stability with no decreaseobserved in either VCC or % live. There is a slight offset in % liveobserved between fresh and frozen material at the lower VCC levels whichis consistent with prior observations that increased VCC is associatedwith better recoveries post-lyophilization.

TABLE 28 VCC and % Live for WP7, Cycle 4 Stored at 30° C. Sample CFU/mL% Live 2-8° C. Stored DS F1300 PreLyo 1.86E+10 96.6 T = 0 h 1.55E+1094.7 T = 24 h 1.53E+10 94.7 T = 48 h 1.62E+10 94.5 T = 72 h 1.53E+1094.3 F0085 PreLyo 1.38E+09 93.3 T = 0 h 1.40E+09 95.7 T = 24 h 1.32E+0995.7 T = 48 h 1.37E+09 94.1 T = 72 h 1.33E+09 94.2 Frozen DS F1300PreLyo 1.70E+10 96.1 T = 0 h 1.52E+10 93.4 T = 24 h 1.46E+10 93.9 T = 48h 1.40E+10 93.7 T = 72 h 1.48E+10 93.4 F0085 PreLyo 1.28E+09 90.1 T = 0h 1.24E+09 94.0 T = 24 h 1.19E+09 92.9 T = 48 h 1.19E+09 90.9 T = 72 h1.18E+09 90.85.12 Conclusion

Lyophilization runs with non-frozen and frozen DS were performed. Twotarget bacterial concentrations were tested. No annealing step or holdwas included during freezing, as there were no advantages during theprevious cycles.

The optical appearance of the lyophilization cake of all formulations(two different bacterial concentrations, frozen and unfrozen rawmaterial) was good. The shrinkage of the lyophilization cake appeared tobe dependent on the bacterial concentration. Less shrinkage was observedfor a higher bacterial concentration.

Reconstitution time was dependent on the bacterial concentration and onthe freezing step. Longer reconstitution times were observed for ahigher bacterial concentration. Shorter reconstitution times wereobserved in these experiments, which did not have an annealing stepduring freezing, compared to experiments having an annealing step duringfreezing (data not shown).

The number of subvisible and submicron particles was mainly unchangedafter lyophilization and storage at 30° C. (analyzed by RMM and MFI).The number of particles of the non-frozen material was higher beforelyophilization. After lyophilization, a small particle population with asize of about 300 nm was detected (analyzed by RMM).

A decrease of the plate based VCC to about 60-70% (relative to Tliq) wasobserved after lyophilization for the lowest bacterial concentration. Ahigher VCC (70-78% relative to Tliq) was observed for the two higherbacterial concentrations. A further decrease of 10-20% was observedafter storage for up to 72 h at 30° C. Storage of the DS in a 1 L LDPEbag with thawing at 37° C. demonstrated comparable VCC and % liveresults on accelerated stability to the DS that was continuouslyprocessed.

Flow Cytometry Analysis for VCC and % live demonstrated good stabilityfor both the frozen and fresh DS. There was a slight offset for thelower VCC level between the fresh and frozen DS.

A RM of about 3% (higher bacterial concentration) and 3.5% (lowerbacterial concentration) was obtained after lyophilization (SDtemperature of 0° C. for 6 h).

In general, no differences were observed between the frozen and thenon-frozen material with the applied methods, indicating that long-termstorage of the DS at −80° C. may be possible, thereby eliminating theneed for continuous manufacturing.

Example 6. Effect of Temperature and Time in Thawing Frozen DrugSubstance Prior to Lyophilization

Temperature and the time for thawing can impact stability. Identifyingappropriate conditions for thawing frozen drug substance allows freezingand holding of the drug substance prior to lyophilization. Ensuringhigh-quality healthy cells coming out of thaw ensures that the resultinglyophilized drug product is also of sufficient quality.

6.1 Freeze-Thawing (FT) of the Bulk Drug Substance

Throughout development, various studies evaluated the ability to storeDrug Substance (DS) at −80° C., thaw and compound manufacture aLyophilized Drug Product (DP) batch at a later date. This involves afreeze-thaw of the DS. A freeze/thaw cycle was defined as the completethawing of the DS followed by storage at −80° C. for a minimum of 24hours), until no ice crystals remain. The stress from the freeze-thawcould adversely impact the product quality (e.g. VCC, % live) of thelyophilized product. Hence, a series of experiments were performed todetermine the optimal storage conditions and thawing procedure for theDS.

6.2 Evaluation of DS Container (Bag Vs. Bottle)

Early studies evaluated DS that was stored in bottles (VibalogicsExperiments Lyo8, Lyo12, and Lyo16, and Coriolis WP2A, WP2B, and WP3).Other studies thawed the DS at either 2-8° C. (Vibalogics Lyo8 and Lyo12and Coriolis WP2B and WP3) overnight or 37° C. in a shorter span of time(Vibalogics Lyo16 and Coriolis WP2A, WP6, and WP7-Cycle 1). Laterstudies evaluated DS that was stored in a 1 L LDPE bag and thawed at 37°C. (WP7-Cycle 3).

6.3 Evaluation of DS Thaw Temperatures and DS Concentration

Since storage of the DS in LDPE bags is preferred over storage inbottles for GMP manufacturing, development efforts focused on conditionsthat resulted in higher VCC and % live profiles when stored in bags. Afreeze/thaw cycle was defined as the complete thawing of the DS followedby storage at −80° C. for a minimum of 24 hours. Freeze-thaw studieswere completed to evaluate the Drug Substance VCC and % live over threeFT cycles for a range of DS VCC levels (concentration) and thawingtemperatures.

The FT studies evaluated DS at OD₆₀₀ of 3.5 and 6.5. Approximately 1 Lof DS was filled into 1 L LDPE bags and frozen at −80° C. and subjectedto three FT cycles at either 2-8° C., Room Temperature (RT) or 37° C. DSbags, at an OD₆₀₀ of 6.5 or at an OD₆₀₀ 3.5, were thawed in a 4° C.refrigerator for 36 hours, at room temperature on a laboratory bench forapproximately 12 hours or at 37° C. in an incubator for ≤8 hours. Afterthawing completely, a sample was taken for analysis and the bags wereplaced in −80° C. for a minimum of 24 hours prior to thawing at therespective conditions and re-freezing for the next cycle.

For the studied thawing conditions, VCC and viability values of BDS atan OD₆₀₀ of 6.5 demonstrated a higher VCC and viability over the threefreeze-thaw cycles relative to the BDS at an OD600 of 3.5 as shown inFIG. 64 and FIG. 65.

The viability for the BDS at an OD₆₀₀ of 3.5 decreased with multiplefreeze thaw cycles for all thaw temperatures except the 37° C. thaw. The37° C. thaw demonstrated freeze-thaw stability for both OD₆₀₀ for 6.5and 3.0.

The data demonstrate that thawing the DS at 37° C. results in improvedproduct quality over the range of VCC values evaluated. The data arefurther supported by the Coriolis experiments which evaluated DS storedas a concentrated pellet, in a bottle and thawed at 2-8° C. which didnot result in an acceptable accelerated stability profile. Based on thefindings WP7 cycle 3 was performed to evaluate if the target storage andthaw conditions of the DS led to an improved in Lyo DP stabilityrelative to DS that had not undergone a FT cycle. The results ofWP7-cycle 3 which compared 2-8° C. and −80° C. stored DS with 1 L fillin a 1 L LDPE bag thawed at 37° C. demonstrated comparable resultsdemonstrating that it is feasible to freeze and thaw DS prior tolyophilization.

Example 7. Exemplary Lyophilization Conditions

7.1 Formulation

TABLE 29 Exemplary Formulation. Target Buffer Composition for LYOFormulation Component Weight of Weights component Molarity MolarityComponents (g/L) (g/mol) (mol/L) (millimol/L) Potassium  0.20 136.080.001470 1.5 dihydrogen phosphate (KH₂PO₄) Disodium 1.14-1.15 141.960.008030 8.0 hydrogen phosphate (Na₂HPO₄) Sucrose 25.00 342.30 0.07303673.0 Water for Q.S to 1 L Injection (WFI) The Drug Product isreconstituted with normal saline.7.2 Drug Substance Manufacturing and Thawing

The DS manufacturing consists of a single-use closed system of a 20 Lculture bag for fermentation, a tangential flow filtration (TFF)manifold for concentration and buffer exchange and a container manifoldfor DS filling. DS can either be held at 2-8° C. for up to three days orfrozen at −80° C. prior to compounding, filling, and lyophilization. DStarget concentration 3.5-6.5 OD₆₀₀. Thawing of the DS is performed at37° C. in ≤8 hours.

7.3 Lyophilization Cycle

TABLE 30 Exemplary Lyophilization Cycle. Temp, T_(s) Duration of ShelvesVacuum Step Phase Item [hh:mm] [° C.] [mbar] NA Preparation Warm up ofLyo, placement of NA 4 Off temperature and Rx sensors 1 Load Loading ofShelves NA 4 Off 2 Ramp Freezing Ramp samples to −4° C. 00:49 −45 Off(1.0° C./min) 3 Hold Hold at −45° C. 02:00 −45 Off 4 Ramp PrimaryVacuum* 00:01 −45 0.09 5 Hold Drying Vacuum* 00:15 −45 0.09 6 RampHeating Ramp (1.0° C./min) 00:27 −18 0.09 7 Hold Stable ShelfTemp >tbc** −18 0.09 9 Ramp Secondary Heating Ramp (0.2° C./min) 01:30 00.09 9 Hold Drying Stable Shelf Temp 06:00 0 0.09 10  Hold Hold at 0° C.until unloading NA 0  1.0*** 10a Ventilation and manual vial NA 0 500mbar closure (N₂ sparging) *Pirani vacuum sensor is the processcontrolling probe **End of primary drying is defined here as 14 hoursafter the T_(P) P100 probe for the cold spots(s) has crossed the T_(s)set-point of 18° C. ***Only done to avoid vacuum pull after the cycle isfinished during the night7.4 Residual Moisture Target

The residual moisture target is >3.0% at release.

7.4 ADXS DS Platform Process Description

Fermentation is carried out within a single-use closed system providedby rocking wave motion bioreactor technology. The single-use closedsystem consists of a product culture bag for fermentation, a tangentialflow filtration (TFF) system for concentration and buffer exchange, anda product manifold for drug substance container filling. Each of thesecomponents are sterilized by gamma irradiation and received inaccordance with site quality systems.

The platform uses the rocking wave motion technology for fermentation.This technology offers the ability to control the entire processingoperation in a closed system. The bulk DS is harvested by TFF using asingle-use hollow fiber module and single-use disposable filtrationpath.

The composition of the fermentation media and the pH control solution (1M sodium hydroxide) are provided in Table 31. The media for the inoculumis sterile-filtered through two 0.2 μm filters in series into a sterile1 L glass bottle. The fermentation media is sterile-filtered through two0.2 μm filters in series into a sterile 10 L glass bottle.

TABLE 31 Fermentation Media Formulation Table. Formulation ComponentsComponent Weights Fermentation Media Tryptic Soy Broth 1 kg (1 kg) D (+)Glucose 11.11 g (monohydrate) pH Control Solution Sodium Hydroxide 40 g(1M NaOH) pellets WFI 1000 g

The culture bag is pre-connected with probes for dissolved oxygen (DO)and pH monitoring. It is then aseptically filled with 5 L offermentation medium. The culture bag is inflated with 0.2 μm filteredcompressed O₂ and air.

Filtered (0.2 μm) compressed air/O₂ is continuously fed duringpropagation at a rate of 1.0 L/minute, the O₂ flow set point is 50%, andis removed through a vent port. The rocking angle is set at 10°. The DOcontrol is set to speed with a rocking rate between 18-36 rpm. The pHcontrol bottle is aseptically connected to the culture bag. Duringpropagation, the process is automatically monitored and controlled fortemperature, pH and dissolved oxygen by an integrated controllingsystem.

A pre-culture is initiated from the working cell bank by pipetting 1 mLof the WCB into 170 mL of fermentation media and grown for approximately10 hours until an OD₆₀₀ of ≥3.5 is reached. The pre-culture is used toinoculate the production culture by aseptically transferring it to theculture bag. Growth is proceeded to an OD₆₀₀≥7.5. When the OD₆₀₀ reachesthe target value, the culture bag is connected, using Ready Mateconnectors to the sterile TFF manifold for concentration anddiafiltration against the formulation buffer. The TFF module uses a 0.2μm pore size hollow fiber filter, meeting the low shear requirements ofcell separation applications. A peristaltic pump is used to feed thefermentation culture into the TFF system. The bulk culture in therecirculation loop is initially set to a flow rate of approximately 75rpm (approximately 4.5 L/min). The fermentation broth is concentrated5-fold to a mass of approximately 1000 g. A permeate pump is used andset initially at 20% (approximately 275 mL/min).

The diafiltration/washing of the harvest concentrate is performed with≥7 diavolumes. The retentate drug substance is sampled from the TFFassembly using an in-process sampling manifold. The OD₆₀₀ of the sampleis measured and used to calculate the dilution volume needed to reach atarget OD₆₀₀ of ≤6.5. The required amount of formulation buffer ispumped into the retentate bag to dilute the retentate to the requiredconcentration. All volume transfers are controlled by weight change inthe respective bags in addition to the complete TFF assembly to controlvolume transfers. The retentate is sampled and measured to confirm theOD₆₀₀ is ≤6.5. If the OD₆₀₀ has not been sufficiently diluted, it may befurther diluted. The DS is then distributed into approximately 1 Laliquots into product bags.

Each bag is heat-sealed for removal from the assembly. Each bag isindividually labeled with the appropriate information and then stored at−70±15° C.

7.6 Drug Substance Process Flow Diagram

See FIG. 20.

7.7 Drug Product Process Flow Diagram

See FIG. 21.

We claim:
 1. A method for producing a lyophilized composition comprisinga Listeria strain, comprising: (a) providing a composition comprising aListeria strain in a formulation comprising a buffer and sucrose; (b)cooling the composition provided in step (a) at a holding temperaturebetween about −32° C. and about −80° C. in a freezing step; (c) exposingthe composition produced by step (b) to a vacuum at a holdingtemperature between about −10° C. and about −30° C. in a primary dryingstep; and (d) exposing the composition produced by step (c) to a vacuumat a holding temperature between about −5° C. and about 25° C. in asecondary drying step, whereby the lyophilized composition is produced.2. The method of claim 1, wherein prior to step (a), a stress responseis induced in the Listeria strain by exposing the Listeria strain to adecreased temperature.
 3. The method of claim 1, wherein prior to step(a), a stress response is not induced in the Listeria strain by exposingthe Listeria strain to a decreased temperature.
 4. The method of claim1, wherein the Listeria strain used in the composition in step (a) is afrozen Listeria strain that is thawed prior to step (a).
 5. The methodof claim 4, wherein the concentration of the frozen Listeria strainbeing thawed is between about 1x10E9 to about 1x10E10 colony formingunits (CFU) per milliliter and/or wherein the formulation comprisesabout 1x10E9 to about 1x10E10 colony forming units (CFU) of Listeria permilliliter.
 6. The method of claim 4, wherein the frozen Listeria strainis thawed at about 2° C. to about 37° C., about 20° C. to about 37° C.,about 32° C. to about 37° C., or about 37° C.
 7. The method of claim 4,wherein the frozen Listeria strain is thawed for no more than 8 hoursand/or wherein the frozen Listeria strain is held at about 2° C. toabout 8° C. for no more than 24 hours after thawing.
 8. The method ofclaim 1, wherein the Listeria strain used in the composition in step (a)is freshly cultured prior to step (a).
 9. The method of claim 1, whereinthe buffer is a phosphate buffer.
 10. The method of claim 1, wherein theformulation comprises about 1% to about 5% w/v sucrose, about 2% toabout 3% w/v sucrose, or about 2.5% w/v sucrose.
 11. The method of claim1, wherein the formulation does not comprise one or more of trehalose,monosodium glutamate (MSG), and recombinant human serum albumin (rHSA).12. The method of claim 1, wherein the holding temperature in thefreezing step (b) is between about −40° C. and about −50° C. or is about−45° C.
 13. The method of claim 1, wherein the freezing step (b)comprises decreasing the temperature to the holding temperature at arate of about 1° C. per minute, and/or wherein the cooling in thefreezing step (b) is for about 2 hours to about 4 hours, and/or whereinthe cooling in the freezing step (b) comprises holding the compositionat the holding temperature for about 2 hours.
 14. The method of claim 1,wherein the holding temperature in the primary drying step (c) isbetween about −12° C. and about −22° C. is between about −17° C. andabout −19° C., or is about −18° C.
 15. The method of claim 1, whereinthe primary drying step (c) comprises increasing the temperature to theholding temperature at a rate of about 1° C. per minute, and/or whereinthe primary drying step (c) is for about 25 hours to about 35 hours,and/or wherein the end of the primary drying step (c) is about 12 toabout 16 hours after the composition has reached holding temperature,and/or wherein the primary drying step (c) is at a vacuum pressure ofabout 0.09 mbar.
 16. The method of claim 1, wherein the holdingtemperature in the secondary drying step (d) is between about −5° C. andabout 20° C., is between about −5° C. and about 5° C., or is about 0° C.17. The method of claim 1, wherein the secondary drying step (d)comprises increasing the temperature to the holding temperature at arate of about 0.2° C. per minute and/or wherein the secondary dryingstep (d) is for about 1 hour to about 10 hours.
 18. The method of claim1, wherein the secondary drying step (d) comprises holding thecomposition at the holding temperature for about 2 hours to about 6hours or about 5 hours to about 6 hours.
 19. The method of claim 1,wherein the secondary drying step (d) is at a vacuum pressure of about0.09 mbar.
 20. The method of claim 1, wherein the residual moisture inthe lyophilized composition is between about 1% and about 5% is betweenabout 2% and about 4%, is at least about 2.5%, or is at least about 3%.21. The method of claim 1, wherein the lyophilized composition shows atleast about 60% viability after storage at between about −20° C. andabout 4° C. for about 12 months, shows at least about 75% viabilityafter storage at between about −20° C. and about 4° C. for about 12months, or shows at least about 80% viability after storage at betweenabout −20° C. and about 4° C. for about 12 months.
 22. The method ofclaim 1, wherein the Listeria strain is a recombinant Listeriamonocytogenes strain.
 23. The method of claim 1, wherein the Listeriastrain is a recombinant Listeria monocytogenes strain, and wherein thebuffer is a phosphate buffer, and wherein the formulation comprisesabout 2% to about 3% w/v sucrose, and wherein the formulation does notcomprise trehalose, MSG, or rHSA, and wherein the formulation comprisesabout 1×10E9 to about 1×10E10 colony forming units (CFU) of Listeria permilliliter, and wherein the holding temperature in the freezing step (a)is between about −40° C. and about −50° C., and wherein the holdingtemperature in the primary drying step (c) is between −17° C. and −19°C., and wherein the holding temperature in the secondary drying step (d)is between −1° C. and 1° C., and wherein the residual moisture in thelyophilized composition is between about 2.5% and about 4%.
 24. Themethod of 23, wherein the Listeria strain used in the composition instep (a) is a frozen Listeria strain that is thawed prior to step (a),and wherein the concentration of the frozen Listeria strain being thawedis between about 1×10E9 to about 1×10E10 colony forming units (CFU) permilliliter, and wherein the frozen Listeria strain is thawed at about37° C., and wherein the frozen Listeria strain is thawed for no morethan 8 hours, and wherein the frozen Listeria strain is held at about 2°C. to about 8° C. for no more than 24 hours after thawing.
 25. Themethod of claim 1, wherein the Listeria strain is a recombinant Listeriastrain comprising a nucleic acid comprising a first open reading frameencoding a fusion polypeptide, wherein the fusion polypeptide comprisesa PEST-containing peptide fused to a disease-associated antigenicpeptide.
 26. The method of claim 25, wherein the recombinant Listeriastrain is an attenuated Listeria monocytogenes strain comprising adeletion of or inactivating mutation in prfA, wherein the nucleic acidis in an episomal plasmid and comprises a second open reading frameencoding a D133V PrfA mutant protein or wherein the recombinant Listeriastrain is an attenuated Listeria monocytogenes strain comprising adeletion of or inactivating mutation in actA, dal, and dat, wherein thenucleic acid is in an episomal plasmid and comprises a second openreading frame encoding an alanine racemase enzyme or a D-amino acidaminotransferase enzyme, and wherein the PEST-containing peptide is anN-terminal fragment of LLO.